Nanopore device for reversible ion and molecule sensing or migration

ABSTRACT

Disclosed are methods and devices for detection of ion migration and binding, utilizing a nanopipette adapted for use in an electrochemical sensing circuit. The nanopipette may be functionalized on its interior bore with metal chelators for binding and sensing metal ions or other specific binding molecules such as boronic acid for binding and sensing glucose. Such a functionalized nanopipette is comprised in an electrical sensor that detects when the nanopipette selectively and reversibly binds ions or small molecules. Also disclosed is a nanoreactor, comprising a nanopipette, for controlling precipitation in aqueous solutions by voltage-directed ion migration, wherein ions may be directed out of the interior bore by a repulsing charge in the bore.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of application Ser. No. 16/422,735filed May 24, 2019, which is a continuation of 14/641,064 filed Mar. 6,2015, now issued as U.S. Pat. No. 10,345,260, which is a Divisional ofapplication Ser. No. 13/411,221 filed on Mar. 2, 2012, now issued asU.S. Pat. No. 8,980,073, which claims benefit of U.S. Provisional PatentApplication No. 61/449,379, filed on Mar. 4, 2011, and which are herebyincorporated by reference in its entirety.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with Government support under Contract NumberNCC9-165 and NNX08BA47A awarded by the National Aeronautics and SpaceAdministration (NASA), Contract Number P01-HG000205 awarded by theNational Institutes of Health, Contract Number NNX09AQ44A awarded byNASA and under Contract Number U54CA143803 awarded by the NationalCancer Institute. The Government has certain rights in this invention.

REFERENCE TO SEQUENCE LISTING, COMPUTER PROGRAM, OR COMPACT DISK

None.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to the field of nanomaterials andspecifically to nanopore devices and sensors. In particular, theinvention relates to sensing and manipulating and sensing ions andcarbohydrates using ionic current measurements on a nanoscale.

Related Art

Presented below is background information on certain aspects of thepresent invention as they may relate to technical features referred toin the detailed description, but not necessarily described in detail.That is, individual parts or methods used in the present invention maybe described in greater detail in the materials discussed below, whichmaterials may provide further guidance to those skilled in the art formaking or using certain aspects of the present invention as claimed. Thediscussion below should not be construed as an admission as to therelevance of the information to any claims herein or the prior arteffect of the material described.

Nanopore Ionic Current Modulators

Solid state nanopores are of great interest as stable structures thatcan be used to mimic biological channels, for the size-selectivesynthesis of nanoparticles or as nanoscale sensors. Conical, orasymmetric, nanopores are a distinct category of nanochannels thatdisplay voltage-gated ion current and can behave as nanofluidic diodes,i.e. they exhibit ionic current rectification. Several groups havedeveloped electrical sensors utilizing ion current measurements acrossmembranes containing asymmetric nanopores (Harrell, C. C. et al.,Resistive-pulse DNA detection with a conical nanopore sensor. Langmuir22, 10837-10843, doi:10.1021/1a061234k (2006); Kececi, K., et al.Resistive-pulse detection of short dsDNAs using a chemicallyfunctionalized conical nanopore sensor. Nanomedicine 3, 787-796, doi:10.2217/17435889.3.6.787 (2008); Sexton, et al., Developing syntheticconical nanopores for biosensing applications. Mol. Bia Syst. 3,667-685, doi:10.1039/b708725j (2007); Au, M., et al. Biosensing withFunctionalized Single Asymmetric Polymer Nanochannels, Macromol. BioscL10, 28-32, doi:10.1002/mabL200900198 (2010)). Such devices are generallyprepared by a track-etching method. Quartz based nanopores, fabricatedfrom quartz capillaries, exhibit many of the same electrical propertiesbut are rapidly prepared using a laser puller. Quartz conical nanopores,also called nanopipettes, exhibit many properties of other asymmetricnanochannels and are advantageous in that the pore can be manipulatedwith high spatial resolution, a property that has been used to imagecells at the nanoscale.

Investigations with conical nanopores have given rise to new chemicaland electrical phenomena that challenge existing ideas about bulkmaterials. Recently, ion current oscillations were observed withrectifying conical nanopores (2 to 8 nm diameter) in polyethyleneterepthalate (PET) films, and were attributed to dynamic precipitationin the pore caused by voltage-induced concentration of weakly solublesalts. Current oscillations in much larger pores of silicon nitride orborosilicate glass can be generated at the interface of two solventsusing organic molecules with differential solubility. These phenomenaoffer a new way to electrically monitor nonequilibrium events such asprecipitation in real time and at the nanoscale.

Nanopore Sensors

The stability and ability to mimic biological channels makenanopore-based platforms candidates for studying (bio) molecularinteractions. Solid-state nanopores are stable, their diameter can becontrolled through the fabrication process and they can be integratedinto devices and arrays. Furthermore, their surface properties can beeasily tuned by chemical functionalization, allowing the development ofchemical and biochemical responsive nanopores. Nanopore-based sensorshave incorporated receptors including proteins, DNA, aptamers, ligands,and small biomolecules, allowing a variety of analytes to be targeted.Essential to the sensitivity of many solid-state nanopore sensors is theproperty of ion current rectification (ICR), arising from the selectiveinteraction between ions in solution and the surface of a charged,asymmetrically shaped nanochannel, or conical nanopore. Nanomaterialsexhibiting ICR and used as sensors include track-etched nanopores inpolymer membranes and quartz nanopipettes. In either case, a keychallenge is the surface modification with appropriate receptors.

Conical quartz nanopores have also been functionalized for sensingapplications [See, for example, Sa, N., Fu, V. & Baker, I. A.“Reversible Cobalt Ion Binding to lmidazole-Modifled Nanopipettes.” AnalChem., 82, 9663-9666, doi:10.1021/ac102619j (2010); Fu, Y., Tokuhisa, H.& Baker, I. A. “Nanopore DNA sensors based on dendrimer-modifiednanopipettes.” Chem Commun (Comb), 4877-4879, doi:10.1039Jb910511e(2009); Umehara, S., Karhanek, M., Davis, R. W. & Pourmand, N.“Label-free biosensing with functionalized nanopipette probes.”Proceedings of the National Academy of Sciences 106, 4611- 4616,doi:10.1073/pnas.0900306106 (2009); Actis, P., Mak, A. & Pourmand, N.“Functionalized nanopipettes: toward label-free, single cellbiosensors.” Bioanalytical Reviews 1,177-185,doi:10.1007/s12566-010-0013-y (2010); Actis, P., Jejelowo, 0. &Pourmand, N. “Ultrasensitive mycotoxin detection by STING sensors.”Biosensors and Bioelectronics 26, 333-337 (2010)].

To date, the reversible binding of analytes with nanopore sensors hasproven challenging. However, this is a critical issue if such devicesare to be used for applications such as continuous monitoring orrepeated measurements with one sensor. Multiple uses for a single sensorwill also overcome problems in reproducibly producing pores of the samesize, which limits quantitative measurements for many sensors reportedin the literature. For such applications, the nanopipette is a promisingplatform as the sensor tip can be precisely and rapidly manipulatedbetween samples, or within a single sample, with nanoscale precision. Todate, functionalized nanopores responsive to pH have shown the bestproperties in terms of rapidly reversible and selective behavior.Nanopipettes functionalized with imidazole and responding to cobalt ionscan be regenerated by immersion in solution of low pH, reprotonating theligand (Sa, N.; Fu, V.; Baker, L. A., Reversible Cobalt Ion Binding toImidazole-Modified Nanopipettes. Anal. Chem. 2010, 82 (24), 9963-9966).

Transport through nanopores can be modified by a variety of externalstimuli including voltage and pressure (see Lan, W.-J.; Holden, D. A.;White, H. S., Pressure-Dependent Ion Current Rectification inConical-Shaped Glass Nanopores. J. Am. Chem. Soc. 2011, 133 (34),13300-13303.). Simply changing the salt gradient across a nanopore canaffect transport, and this effect was used to focus DNA forresistive-pulse measurements (see Wanunu, M.; Morrison, W.; Rabin, Y.;Grosberg, A. Y.; Meller, A., Electrostatic focusing of unlabelled DNAinto nanoscale pores using a salt gradient. Nat Nano 2010, 5 (2),160-165.). Nanopores can also be engineered to respond to stimuli suchas solvent polarity. This can be achieved with so-called “hairynanopores,” in which the nanopore is decorated with polymers (see Peleg,O.; Tagliazucchi, M.; Kröger, M.; Rabin, Y.; Szleifer, I., MorphologyControl of Hairy Nanopores. ACS Nano 2011, 5 (6), 4737-4747.). Severalartificial nanopores have been engineered for pH-sensitivity usingsurface modification. Conical nanopores have been modified withreceptors for binding other charged species, which likewise modulatecurrent rectification. Targets have included nucleic acids, metal ions,proteins, and small molecules. In the cases of large biomolecules, suchas nucleic acids and proteins, physical blocking of the pore likelyplays a role in addition to modulation of the surface charge. To date,the modulation of current rectification with small, uncharged specieshas proved difficult. However, such a system would expand the stimulifor responsive nanopores to include drugs, peptides, and carbohydrates.

Glucose/Diol Sensing

Carbohydrate recognition is essential to monitoring of blood glucose(Kondepati, et al. Anal. Bioanal. Chem. 388, 545-563 (2007). Detectionand quantification of carbohydrates can also be used in bioprocessmonitoring and for medical diagnostics based on metabolic saccharides,nucleotides, or glycoproteins (Timmer, et al. Curr. Op. Chem. Biol. 11,59-65 (2007). Most electrochemical methods for measuring glucose rely onredox enzymes such as glucose oxidase (Oliver, et al. Diabetic Med. 26,197-210 (2009). The most common artificial receptors use boronic acids,which have predominately been used for optical probes (Mader & Wolfbeis,Microchimica Acta 162, 1-34 (2008). Non-enzymatic methods forelectrochemical measurement of glucose have also been developed, mostlyrelying on oxidation of glucose (Park, et al. Anal. Chim. Acta 556,46-57 (2006); E.T Chen, Nanopore structured electrochemical biosensors,US 2008/0237063).

To date, there has been very little reported in the literature onnanofluidic pores that respond to carbohydrates. Nanopore analytics havebeen used to detect small molecules using resistive-pulse methods, butthe technique is generally more suited to proteins and othermacromolecules. Oligosaccharides on the order of MW 500 to 10,000 havebeen discriminated using resistive-pulse techniques with alpha-hemolysinpores.

One example of receptor-modified nanopores uses with a covalentlyattached HRP enzyme, which is then conjugated supramolecularly to Con A,a saccharide-binding protein which interacts with mannose units on theHRP molecule (Ali, et al. Nanoscale 3, 1894-1903 (2011). Addition ofmonosaccharides (galactose and glucose) competes with the Con A,changing the ion current through the pore. Two recent examples make useof boronic acid as a chemical receptor, where the receptor is attachedcovalently to the walls of artificial nanopores (Sun, Z.; Han, C.; Wen,L.; Tian, D.; Li, H.; Jiang, L., pH gated glucose responsive biomimeticsingle nanochannels. Chem. Commun. (Cambridge, U. K.) 2012.; Nguyen, Q.H.; Ali, M.; Neumann, R.; Ensinger, W., Saccharide/glycoproteinrecognition inside synthetic ion channels modified with boronic acid.Sensors and Actuators B: Chemical 2012, 162 (1), 216-222.). In the caseof the former, an acidic solution was required to reverse the saccharidebinding and restore the signal. In the latter, reversible binding wasnot demonstrated.

Despite many recent advances in nanopore fabrication and surfacechemistry, the work cited above shows that there is a need for newschemes to modulate ion current using carbohydrates as an externalstimulus. This problem may be addressed with new functional materialsthat can interface with nanopores.

Specific Patents and Publications

Karhanek et al. in US Patent Application Publication 2010/0072080,published on March 25, 2010, disclose methods and devices comprising ananopipette having thereon peptide ligands for biomolecular detection,including of peptides and proteins.

Siwy et al. in U.S. Pat. No. 7,708,871, issued on May 4, 2010, disclosean apparatus having a nanodevice for controlling the flow of chargedparticles in an electrolyte. Such apparatus comprises an electrolyticbath container divided by a polymeric membrane foil for controlling theflow of charged particles in an electrolyte.

Sa et al. in Analytical Chemistry 2010, 82 (24), pp 9963-9966 disclosethat quartz nanopipettes modified with an imidazole-terminated silanerespond to metal ions (Co²⁺) in solution. The response of nanopipetteswas evaluated through examination of the ion current rectificationratio. When nanopipettes were cycled between solutions of different pH,adsorbed Co²⁺ was released from the nanopipette surface, to regeneratebinding sites of the nanopipette.

Umehara et al. in Proceedings of the National Academy of Sciences, vol106, pages 4611-4616, Mar.24, 2009, disclose a label-free, real-timeprotein assay using functionalized nanopipette electrodes.Electrostatic, biotin-streptavidin, and antibody-antigen interactions onthe nanopipette tip surface were shown to affect ionic current flowingthrough a 50-nm pore.

Umehara et al. “Current Rectification with Poly-L-lysine Coated Quartznanopipettes,” Nano Lett. 6(11):2486-2492 (2006) discloses currentresponses of noncoated and Poly-1-lysine coated nanopipettes using ananopipette in a bath solution.

Karhanek M., Kemp J. T., Pourmand N., Davis R. W. and Webb C. D, “SingleDNA molecule detection using nanopipettes and nanoparticles,” Nano Lett.2005 February; 5(2):403-7 discloses that single DNA molecules labeledwith nanoparticles can be detected by blockades of ionic current as theyare translocated through a nanopipette tip formed by a pulled glasscapillary. The disclosed set up uses a voltage clamp circuit, whichutilized a single detecting electrode in a bath to detectnanoparticle-DNA current block.

Ying, Liming in Biochemical Society Transactions, vol 37, pages 702-706,2009, reviews nanopipettes and their use in nanosensing andnanomanipulation of ions, molecules (including biomolecules) and cells.

Borghs, Gustaaf, et al. in WO 2006/000064, published on Jan. 5, 2006,disclose a nanofluidic device for controlling the flow of chargedcarriers through a nanopore extending through a membrane.

Sunkara, et al. in US Patent Application Publication 2005/0260119,published on Nov. 24, 2005, disclose a method of synthesizing tubularcarbon nanostructures in the form of tapered whiskers, termednanopipettes, using microwave plasma assisted chemical vapor depositionmethod.

Chen US 20080237063 published Oct. 2, 2008, entitled “Nanoporestructured electrochemical biosensors,” discloses a biosensor having ananopore structured and catalytically active cyclodextrin attachedthereto for direct measurement of glucose.

Choi et al., “Biosensing with conically shaped nanopores and nanotubes,”Phys. Chem. Chem. Phys. 8:4976-4988 (2006) discusses the preparation andcharacterization of conical nanopores synthesized using a track-etchprocess. The design and function of conical nanopores that can rectifythe ionic current that flows through these pores under an appliedtransmembrane potential is also disclosed.

Li et al., “Development of boronic acid grafted random copolymer sensingfluid for continuous glucose monitoring,” Biomacromolecules10(1):113-118 (2009) discloses biocompatible copolymerspoly(acrylamide-ran-3-acrylamidophenylboronic acid) (PAA-ran-PAAPBA) forviscosity based glucose sensing.

Sun, Z.; Han, C.; Wen, L.; Tian, D.; Li, H.; Jiang, L., pH gated glucoseresponsive biomimetic single nanochannels. Chem. Commun. (Cambridge, U.K.)(2012) describe a track-etched conical nanochannel in polyethyleneteraphthalate (PET) covalently modified with phenylboronic acidreceptors.

Nguyen, Q. H.; Ali, M.; Neumann, R.; Ensinger, W.,Saccharide/glycoprotein recognition inside synthetic ion channelsmodified with boronic acid. Sensors and Actuators B: Chemical 2012, 162(1), 216-222.) describe a track-etched conical nanochannel inpolyethylene teraphthalate (PET) covalently modified with phenylboronicacid receptors. The channel responds to monosaccharides as well asglycoproteins.

BRIEF SUMMARY OF THE INVENTION

The following brief summary is not intended to include all features andaspects of the present invention, nor does it imply that the inventionmust include all features and aspects discussed in this summary.

The present invention, in certain aspects, is directed to a nanopipettefor use in an apparatus detecting an analyte in a sample, comprising acapillary portion defining an interior bore of the nanopipette leadingto a nanopore opening; said interior bore adapted for containing thereinan electrode and adapted to contain an interior solution communicatingwith an exterior solution through said nanopore (see, for electrodeconfiguration, e.g. FIG. 1, where the bore is elongated and tapers tothe opening); and a coating on an interior surface of the nanopore,comprising a polyelectrolyte bound directly to (i.e. contacting) saidinterior surface (typically quartz); and a binding molecule, linked tosaid polyelectrolyte, specific for binding an analyte selected from thegroup consisting of an ion or a small molecule.

In certain aspects of the present invention, the binding molecule linkedto the polyelectrolyte may be a boronic acid for sensing glucose, and/orthe polyelectrolyte may be a polycation whereby the charge of thecoating changes; and/or the polyectrolyte may be polyalkyl pyridine or apolyamine.

In certain aspects, the present invention, the polyelectrolyte/sensingmolecule is applied to the nanopore so as to extend into and partiallyblock the nanopore, exposing more of the sensing molecule to the sample.

In certain aspects of the present invention, the binding molecule is achelating agent linked to a polymer or polyelectrolyte coating. Thepolymer coating may further comprise a polyelectrolyte layer between thepipette bore and the ion binding polymer. The layer may be in the formof a coating, and preferably is continuous, whereby bare quarts iscovered. In some embodiments, the chelating agent may be an ion bindingpolymer which is a polysaccharide, for example, chitosan, a linearpolysaccharide. In some embodiments, the chelating agent may be apolypeptide. In some other embodiments, the coating may comprise asaccharide binding molecule. In some embodiments, the saccharide bindingmolecule may be a protein, such as a lectin. In some embodiments, thesaccharide binding molecule comprises boronic acids or boronic esters.

In a further embodiment, the present invention comprises a nanopipettedevice for sensing carbohydrate molecules, in particular carbohydrateswith cis-diol groups, and, more particularly, glucose. The device maycomprise an inert substrate bearing a nanopore in which the nanoporecomprises a channel. In certain aspects, the channel is a quartznanochannel. In some aspects, the inert substrate defines an interiorportion accessed through the channel and an exterior portion forcontacting with a sample. In some aspects, the nanopore furthercomprises a polymeric coating within the channel, said polymer linked toa carbohydrate-binding molecule (“CBM,” such as boronic acid). In someaspects, the nanopore further comprises a polymer linked to said CBM,wherein the CBM is embedded within the polymer to form a semi-permeablematrix within the channel.

The device is operated with a measuring circuit in which ionic currentrectification by ionic conditions at the nanopore is modulated bybinding of the glucose analyte. In a preferred embodiment, binding of asaccharide to the polymer-linked boronic acid causes a reversal of ionicrectification. Thus, the device may comprise an electronic circuit forproducing a current through the channel and measuring changes in currentflow through the channel, whereby carbohydrate molecules in said samplebind to said CBM, resulting in a measured change in current flowindicative of carbohydrate presence in the sample.

In some aspects, the present invention comprises a nanopipette measuringcircuit comprising a nanopipette having an interior electrode, a borefor holding an interior solution, a container for an exterior solution,and a measuring circuit between the interior electrode and an externalelectrode adapted and located to be in contact with an exteriorsolution, wherein the measuring circuit comprises an amplifier fordelivering a reversible voltage differential between said interiorelectrode relative to the external electrode, and further comprises adetector for measuring current between the bore and the exteriorsolution.

Certain aspects of the invention are concerned with methods for creatingionic compounds by combining two different ions in solution, i.e. ananoscale reactor, comprising steps of providing at least one first ionhaving a charge in a solution inside of a nanopipette with a nanoporebetween an interior of the nanopipette and an exterior solution;providing a second ion species in the exterior solution; and applying tothe one ion having a charge in a solution inside of a nanopipette avoltage across the nanopore of opposite charge, said voltage sufficientto cause migration of said ion species to the nanopore to react withsaid second ion to form said ionic compound. In some embodiments, thechange in current indicative of formation of the ionic compounds may beoscillations.

In certain aspects, the present methods further comprise the step ofmeasuring ionic current through the nanopore and detecting a change incurrent indicative of formation of the ionic compound.

In some embodiments, the ionic compounds are insoluble. The method mayfurther comprise the step of reversing the voltage after a precipitatehas formed.

The first or second ions may be a metal ion reacting with an anion toform the ionic compound. The metal may be a transition metal. The metalion from the group consisting of Mg2+, Ca2+, Mn2+, Zn2+, Cu2+, Fe2+,Fe3+, Cr3+, Cr6+, Cd2+, Mo2+, Co3+, Co2+, Hg2+, Ni2+, Al3+, Al2+, Ar3+,Ar3− and Pb2+. The anion may be from the group consisting of phosphate,chloride, sulfate, monophosphate, pyrophosphate, metaphosphate,tripolyphosphate, tetrametaphosphate, and orthophosphate. In someembodiments, the anion may be selected from the group consisting oforganic carboxylic acid anions selected from the group consisting ofgluconate, tartrate, fumarate, maleate, malonate, malate, lactate,citrate, EDTA, citraconate, citramalate, stearate, oleate, laurate,octoate ascorbate, picolinate, and orotate. In some other embodiments,the anion may be a protein.

In certain aspects, the present invention is directed to a method ofdelivering an ion into an exterior solution from inside a nanopipettecomprising providing at least one first ion having a charge in asolution inside of a nanopipette with a nanopore between an interior ofthe nanopipette and an exterior solution; and applying a voltage acrossthe nanopore of opposite charge to said first ion, said voltagesufficient to cause migration of the ion across the nanopore.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the electrochemical setup andthe reversible binding of cupric ions on a sensor that has beenfunctionalized to specifically bind such ions in the vicinity of theinterior of the nanopipette, near the tip.

FIG. 2 is a schematic representation of an electrochemical set up as inFIG. 1, where formation of a precipitate at the nanopore causes ameasurable blockage of ionic current.

FIG. 3 is a graph that shows current oscillations in a nanopipette setupused for measuring precipitation, with 2 micromolar zinc chloride in thebath and a potential of −350 mV. Inset: expanded view of one of the openstates.

FIG. 4 is an IV plot showing data from an ion-binding surfacemodification (calmodulin/PAA/PLL) that would be applied as shown at 108in FIG. 1, and shows ion sensitivity of the nanopipette biosensor. Solidline-bare pipette; dashed line-PAA2; triangles CaM.

FIG. 5A is a graph that shows the rectification coefficient for surfacesof sensor CaM-1.

FIG. 5B is a current trace of the sensor of FIG. 5A.

FIG. 6 is a graph that shows the pH response of a bare nanopipette(triangles) and chitosan/PAA improved nanopipette sensors (circles).

FIG. 7 is a graph showing variation of the rectification coefficient vs.numbers of chitosan/PAA layers deposited at pH 3 and 7.

FIG. 8 is a graph that shows the pH response of a bare nanopipette(triangles) and chitosan/PAA sensor (circles).

FIG. 9 is a graph that shows ion current under various conditions,indicating the effect of functionalization of a sensor with chitosan in0.1 M KCl, 10 mM phosphate buffer solution.

FIG. 10 is a graph showing variation of the rectification coefficientafter recycling of the sensor. Cu2+ concentration: 100 uM. The sensorwas regenerated by immersing the sensor into a pH3 solution for 60seconds.

FIG. 11 is a trace that shows the response of the sensor to variousconcentrations of Cu2+ in 0.1 M KCl, 10 mM Tris-HCl, pH 7. A linear fitbetween 1/I_(n) and 1/C_(copper) (not shown) was calculated (R=0.997).Ion currents were determined at a potential of −500 mV applied to theelectrode in the nanopipette barrel.

FIG. 12 is a cartoon depicting the role of electrophoresis on theinteraction of cupric ions with a nanopipette.

FIG. 13 is a trace showing output current, the arrow indicates theaddition of Cu2+ ions (final concentration in solution 150 uM). Nochange is detected while applying a positive voltage while an immediateresponse occurs upon switching to a negative potential that causes avariation on the following positive step.

FIG. 14 is a cartoon illustration of oscillations in ion current byclearing of a precipitate from a nanopore at a tip of a nanopipette.

FIG. 15A is a graph of oscillations on applying a negative potential toa pipette filled with phosphate buffer (pH 7) and immersed in a bath ofTris-HCl buffer (pH 7) with 2 micromolar zinc chloride.

FIG. 15B is a histogram showing events from FIG. 15A in the states ofhigh and low conductance.

FIG. 16 is a diagram showing the synthesis of a cationic polyelectrolytefor modification of a nanopipette. The polycation is represented byrepeating pyridyl units on an alkyl backbone.

FIG. 17 is a schematic showing direct two-step functionalization of aquartz surface 172 with a boronic acid.

FIG. 18A, 18B and 18C is a series of drawings showing an end view of ananopore and illustrating three methods for immobilization of receptorsto a nanopore. FIG. 18A shows covalent attachment of receptors directlyto the nanopore wall. FIG. 18B shows adsorption of a functional polymerto the pore wall, with receptors linked to the polymer. FIG. 18C showsImmobilization of a three-dimensional functional matrix inside the pore,where receptors on the polymer form a mesh-like network across thenanopore, ie. the coating extends into and partially blocks saidnanopore.

FIG. 19A, 19B and 19C is a set of graphs showing the response of controland functionalized pipette with glucose (3 mM) in pH 7 phosphate buffer.The pipette was functionalized with 3-aminopropyl triethyxysilanefollowed by m-bromomethylphenylboronic acid. Inset: rectificationcoefficients calculated from the ion current at +500 and −500 mV. FIG.19A is a graph showing ion current response of an unmodified pipette;FIG. 19B is a graph showing ion current response of a boronicacid-modified pipette and FIG. 19C is a graph showing rectification ofthe pipette systems shown in FIG. 19A and B.

FIG. 20 is a bar graph showing the precipitation of PVP-BA in thepresence of monosaccharides. A stirring solution of PVP-BA inmethanol/water at pH 2 was titrated with sodium hydroxide untilprecipitation occurred. The error bars show standard deviation fromthree separate experiments.

FIG. 21 is a graph showing the change in absorbance of ARS in thepresence of polymers PVP-BA and PVP-Bn at 1 micromolar polymerconcentration. The dye solution is 0.25 mM in 1:1 methanol/water.

FIG. 22 is an IV plot showing the modulation of ion permeability in thepresence of ARS. A nanopipette embedded with the polymer PVP-BA wasimmersed in pH 9.5 carbonate buffer containing either 0, 60, or 360 μMARS. Error bars shown standard deviation from repeated voltage ramps(N=5).

FIG. 23 is a graph showing the inversion of ion current rectificationwith fructose. Current-voltage curves for a PVP-BA-embedded nanochannelat pH 9.5 without and with 10 mM fructose.

FIG. 24 is a graph that shows sequential cycles of ion currentmodulation measured at −0.5 V with and without fructose. Error bars showthe standard deviation from repeated voltage scans (N=5). Equilibrationtime was 5 minutes at each condition

FIG. 25 is a graphic showing the reversible ion current rectificationvalues for three separate nanopipette sensors. Sensors were immersed incarbonate buffer (pH 9.5) with or without fructose (10 mM). Positiverectification for sensor 51 in buffer is shown at 254. Negativerectification of sensor 51 in fructose is shown at 252.

FIG. 26 is line graph showing binding isotherms for fructose usingPVP-BA in a solution-phase fluorescence assay.

FIG. 27 is a graph of data as shown in FIG. 26 using an electrochemicalmethod using a modified conical nanochannel. FIGS. 26 and 27 wereenerated by titrations of fructose into a probe solution containingPVP-BA (0.003% w/v) and HPTS (1.5 μM) in pH 9.5 carbonate buffer with25% methanol. Error bars show the standard deviation from three separatetitrations. Graph B shows the ion current at potential −500 mV forconical nanochannel embedded with PVP-BA with increasing concentrationsof fructose in a carbonate buffer. Error bars show standard deviationfrom 5 sequential voltage scans. Data points were fitted to bindingisotherm as described in the experimental section.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Overview

The present invention relates to a nanopore device, i.e. a nanopipette,which is comprised in an electrochemical sensor. The device isconfigured to contain an interior region that contains a firstelectrode, and to be contacted with an external solution that containsanother electrode. These electrodes are connected to a sensing circuitwhere phenomena occurring at the nanopore may be detected and measured.In the preferred embodiment, the nanopore is part of a nanopipette, asdescribed for example in the above referenced US 2010/0072080 from thesame assignee. The term “nano” refers to dimensions within the bore, andthe accompanying parts, such as the interior electrode, which will havea diameter on the order of <200 nm. The present dimensions are importantfor creating the electrochemical behavior described in detail below.

The present invention further relates to a reversible ion sensor thatcan bind and detect the binding of small ions such as H+ and metalcations, as well as diols (discussed below). The reversible sensor isreversible in terms of reversing polarity within the sensor cavity (i.e.nanopipette bore) and, importantly, in that the ions are bound to thenanopore in a reversible manner. The present device can be used to causemigration of ions within the sensor and out of the sensor nanopore,which sensor preferably is in the form of a nanopipette. The binding ofions within the pipette can be reversed by immersion of the pipette tipin a solution free of the bound ion. Binding of the small ions can alsobe released by changing conditions such as pH in the solution. Thepresent invention also relates to a reversible ion migration devicebased on a nanopipette. Such ion migration can be induced by changes involtage potential inside the nanopipette relative to the externalsolution. Ion migration can be used to induce precipitation caused bymigration of ions out of the nanopipette bore to create excess ions inthe external solution. The precipitation occurs in the vicinity of thenanopore tip of the nanopipette and can be detected at an early stage bychanges in current oscillations through the nanopore at the tip. Theprecipitation causes changes in an ion current through the nanopore tipinduced by an applied voltage potential between the interior and theexternal solution. The precipitation that is detected at small ionconcentrations and at small precipitate sizes can be applied to avariety of systems. For example, protein crystallization can be detectedusing small amounts of protein.

The present sensor technology relies on a simple electrochemical readoutthat can transduce, in a label-free manner, binding events at the tip ofthe functionalized nanopipette. The high impedance of the nanopipettetip confines the sensitivity of the device, making the dimension andgeometry of the tip orifice crucial for the sensor performance.Furthermore, the present sensor technology can be easily integrated withpiezoactuators to generate a sensor with high spatial resolution. As ananopipette approaches a surface, the ionic current through the pipettewill decrease due to “current squeezing”, a well known effect, exploitedto great benefit in scanning ion conductance microscopy (SICM). Besidessensing, nanopipette based platforms have been used to investigatesingle-molecule biophysics, for the controlled delivery of moleculesinside a single cell, and to image cells at the nanoscale.

In certain embodiments, the present devices present a new mechanism forinducing and/or measuring current oscillations using precipitation in asolid-state nanopore. The technique can be used to actively direct ionmigration to the interface of two solutions at the tip of a nanopipette.Because the pore becomes blocked only at a threshold potential, thereaction can be controlled temporally as well as spatially. Suchprecipitation can be carried out with a variety of ions. This ability tocontrol and measure the kinetics of salt precipitation at the nanoscaleenables new techniques for studying dynamic processes such asbiomineralization and dissolution. The trapping of a precipitate withinthe nanoreactor by voltage oscillations is a further tool to study sizeand surface charge of nanoparticles. The controlled nanoprecipitation ofinsoluble salts is also valuable in developing selective and sensitiveion sensors. The reactions described below were able to detect as littleas 2 micromolar salt, and were unaffected by the presence of othercations. For example, a low concentration (up to 2 μM) of zinc sulphatesalt was detected and was unaffected by the presence of other cationssuch as potassium or magnesium. Furthermore, the ability for a nanoporeblocked by precipitation to be opened through oscillating potentials canexpand the applications for sensing with nanopores. For example, aconstant voltage may be used to detect nanoprecipitates, whileoscillating potentials can be used to measure ion current for othersensing applications.

The present methods may be used in a variety of protein crystallizationmethods. Different crystallization methods are used to bring a proteinsolution into supersaturation, normally through a gradual decrease ofsolubility of the protein. The most common way to reduce proteinsolubility in protein crystallization is by the addition of precipitantssuch as polyethylene glycol and ammonium sulfate. The precipitant bindswater as its concentration is increased, for example by using the methodof vapor diffusion. As a result, the amount of solvent available for theprotein is decreased, which essentially means that the concentration ofthe protein is increased. At a certain effective protein concentration,it will begin to precipitate, creating a crystal if conditions arecorrect. The correct crystallization conditions, which include acombination of a right pH, ionic strength, temperature, proteinconcentration, the presence of various salts, ligands or additives, thetype of precipitant and the actual crystallization method (hanging drop,sitting drop, dialysis, etc.), are practically impossible to predict inadvance; as a result, crystallization screens using different conditionshave been developed. The present device may be employed in a number ofparallel experiments to test the effect of different proteincrystallization conditions.

Another application of the present method is in the field of analyticalion sensing. For what is believed to be the first time, the analyticalapplication of chemically functionalized solid-state nanopores is shownfor ion sensing. The frequency and the waveform of the applied voltagewere found to be adjustable to maximize the signal-to-noise ratio,showing that the applied voltage could trigger the Cu²⁺ binding on thesensor. The ability to temporally and spatially direct the binding ofmolecules allows for the development of precise biosensing devicescapable of studying thermodynamic and kinetic properties of theanalyte-receptor interaction.

The biosensors disclosed herein show selective and reversible binding ina rectifying nanopore. This type of reversible sensor overcomes thechallenges associated with making nanopores of unified pore structuresin ICR-based sensors. Monitoring the deposition of polyelectrolytelayers effectively ensures that farther chemical modifications arelocalized to the pore where ICR is the most sensitive. Furtherexperiments and theoretical modeling, as well as advanced methods ofcharacterizing nanopore surfaces will be required to determine how farinto the pore the polyelectrolytes penetrate and further exploreinteractions at the interface of bulk solutions and the outernanopipette pore. Progress in this area will advance the use of nanoporesensors as analytical tools. Reversible nanopipette sensors such asthose described here may be used for monitoring of water quality,spatial resolution of ion concentration at the nanoscale (functionalmapping) or continuous intracellular measurements of specific analytes.

The present devices have been adapted for glucose sensing as may beneeded by subjects at risk for diabetes. They can be built as portabledevices that can be applied to whole blood from or even in a subject inneed of glucose monitoring. That is, for example, quartz nanopipettetips can penetrate the skin to contact glucose in body fluid in thevicinity of the dermis.

The present glucose-sensing devices preferably employ a polymer which isbound to a receptor such as boronic acid (a receptor for saccharides)and the mixture applied to the interior surface of the nanopipette, ator near the tip (nanopore). The saccharide-binding polymer mixture mostpreferably has the following properties: 1) The polymer is positivelycharged so as to increase interaction with the negatively charged porewalls. 2) The polymer will have approximately one boronic acid for eachpositive charge, resulting in a neutralization of the polymer onconversion to boronate form. 3) The polymer is soluble in organicsolvents but insoluble in aqueous media, such that the polymer can betrapped in the tip of the nanopipette. 4) The insoluble matrix formed bythe polymer is permeable to water, ions, and analytes. 5) No change insolvent or media should be required to reverse signal modulation in thesensor.

The permeable matrix method is thought to be superior to other nanoporefunctionalization methods because the entire volume of the nanopore,rather than only the pore walls, contains the receptor. Thepolymer-bound receptors (e.g. boronic acid) will have greaterinteraction with ions travelling through the nanopore, and thus givehigher signal modulation in the presence of the analyte. Furthermore,many polymers undergo changes in conformation upon analyte binding,which may further enhance ion current modulation. Finally, it isimportant to note that the analyte does not need to be driven throughthe pore by an electric field—rather, passive diffusion of the analytethrough the polymer matrix will modulate the electrical signal. Thesefeatures distinguish an immobilized polymer network from covalentattachment of a receptor or layer-by-layer deposition of functionalizedpolymers. A system based on poly-(4-vinylpyridine) (PVP) was chosen, asdescribed below.

As described in detail below, a nanopipette device was fabricated inwhich functional polycation containing boronic acid glucose receptorswere embedded in a quartz nanochannel. Previous efforts to directlymodify the walls of nanochannels (i.e. nanopores) with boronic acidshave resulted in modest response to saccharides, but there are severalaspects of boronic acid-based receptors that should offer much greatercontrol over ionic current. First, the binding of boronic acids tocarbohydrates is completely reversible, and there is no evidence thatthis is the case in a nanochannel. More importantly, boronic acids canundergo a change in electrostatic charge in the presence of neutralcarbohydrates—a change which should dramatically affect currentrectification in a nanopore. To take advantage of these properties foran engineered nanochannel, a cationic polymer based onpoly(4-vinylpyridine) was chosen as a boronic acid receptor matrix (FIG.26). Alkylation of the polymer produces exactly one boronic acid forevery positive charge. The cationic polymer can be immobilized to thequartz nanopipette based on electrostatic interaction, an interactionthat can be monitored based on current rectification.

Boronic acids as used here are simple artificial receptors that havebeen recognized for their ability to bind saccharides. There are severalproperties of the boronic acid that can be exploited to make sensors andactuators. On binding 1,2-diols (e.g. 1,2 dihydroxy benzene, ethyleneglycol), the Lewis acidity of the boronic acid is increased. Forexample, phenyl boronic acid forms a complex with catechol (1,2benzenediol) in equilibrium with negatively charged boron species (See,Artificial Receptors for Chemical Sensors, edited by Vladimir M. Mirsky,Anatoly Yatsimirsky, Wiley-CH, Chapter 6 (2011)). Thus, if the pK_(a) isshifted to a value lower than the pH of the buffering medium, binding ofa carbohydrate results in conversion of the boronic acid to the anionicboronate ester. Many of the probes and sensors reported to date usingboronic acids are fluorescence-based. Because of the versatility ofboronic acids, however, this receptor has also been used forcarbohydrate separations, optical sensors based on swelling of polymericmaterials, and electrochemical sensors. The fact that binding a neutralsaccharide can effect a change in the charge of boronic acids makes thisreceptor an excellent candidate for engineering responsive nanofluidicdiodes.

Modified nanopores within nanochannels showed strong currentrectification resulting from the cationic charge of the polymer, arectification that can be inverted in the presence of neutralcarbohydrates. The nanochannels showed reversible behavior withmillimolar concentrations of fructose. The ability to characterize thepolymer in solution showed a binding mode dependent both on theinteraction of 1,2-diols with boronic acids, as well as electrostaticcharge. Modified nanochannels showed especially high sensitivity to theanionic catechol-containing dye, alizarin-red sulfonate (ARS), withcancellation of current rectification using only 60 μM dye. Importantly,the modulation of ion permeability relied not on blocking ionconductance, but in changing the polarity of current rectification as aresult of electrostatic charge. This application will enable newtechniques independent of voltage for modulating ion flow throughnanochannels.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. Generally, nomenclatures utilized inconnection with, and techniques of, cell and molecular biology andchemistry are those well known and commonly used in the art. Certainexperimental techniques, not specifically defined, are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. For purposes of theclarity, following terms are defined below.

The term “ion” means an atom or molecule in which the total number ofelectrons is not equal to the total number of protons, giving it a netpositive or negative charge, The term refers specifically to an atomicor a monoatomic ion, where the ion consists of a single atom, and topolyatomic ions of small molecules.

The term “small molecule” means a compound having a molecular weight ofless than about 1,000 atomic mass units, or, in some embodiments lessthan 200 amu. Small molecules do not include polynucleic acids orpolypeptides above the size limit, but include other small moleculestypically found in a cell. By way of example, adenine is about 135 amu;glucose is about 180 amu; urea is about 60 amu; creatinine is about 113amu.

The term “nanopipette” means a hollow self-supporting, inert,non-biological structure with a conical tip opening of nanoscale, i.e.,a nanopore, having a tip opening of 0.05 nm to about 500 nm, preferablyabout (+ or −20%) 50nm or about 80 nm or about 100 nm. The hollowstructure may be e.g. glass or quartz, and is suitable for holdinginside of it a fluid which is passed through the tip opening. Theinterior of the nanopipette is selected or modified to minimizenonspecific binding of analyte. The interior of a nanopipette typicallyis in the form of an elongated cone, with a uniform wall thickness of asingle layer of quartz or other biologically inert material, and issized to allow insertion of an electrode that contacts solution in thenanopipette. The nanopipettes used herein typically have a single bore,but nanopipettes with multiple concentric bores can be prepared bypulling dual bore capillary tubes. The outer diameter is typically lessthan about 1 μm in the tip region.

The term “nanopore” means a small hole in an electrically insulatingmembrane, preferably the tip of a nanopipette, as described. Thenanopore will be in a tip region, which is the last few mm of thenanopipette bore, adjacent the nanopore. The nanopore, as describedbelow, is sized so that small molecular complexes will affect movementof ions and molecules through the nanopore. The nanopore is designed tofunction in a device that monitors an ionic current passing through thenanopore as a voltage is applied across the membrane. The nanopore willhave a channel region formed by the nanopipette body, and, preferably,will be of a tapered, e.g. frusto-conical configuration. By pulling aquartz capillary as described below, a reproducible and defined nanoporeshape may be obtained.

The term “current rectification” means an effect when charged nanoporesrespond to a symmetric input voltage with an asymmetric current output.When the diffuse electrical double layer thickness is comparable withthe pore size, the electrostatic interactions between fixed chargedspecies on the nanopore surface and ionic species in solutions altersnanopipette permselectivity. The rectification coefficient, r, isdefined as the logarithm of the ratio between the current measured atparticular positive voltage and the current measured at the same voltagebut with the opposed polarity, i.e. r=Log₁₀ I+/I−.

This coefficient is a useful indicator of the rectifying properties of ananopipette and therefore of the fixed charges on the sensor surface.Quartz nanopores, being negatively charged, show a negative currentrectification (r<0). The rectification can be inverted (r>0) bymodifying the nanopore surface with charged functional layers such aspoly-L-lysine, dendrimers, aminosilane and chitosan.

The term “nanopipette apparatus” means a nanopipette operativelyconnected to a current detecting circuit and adapted to receive a samplefluid in contact with the nanopipette and any reference electrode.

The term “current detecting circuit” means a device for detectingcurrent and/or voltage, and applying same in a circuit, such asconnected to a nanopipette and a reference electrode as describedherein. The circuit may comprise any sensitive device for detectingchanges in current on the order of 1-100, 10-100 or 1-10 picoamperes,based on a baseline current of 10-10000 picoamperes. The term furtherrefers to a circuit that is time responsive and relatively temperatureindependent or allow for changes in temperature to be compensated for.It should have an input in a circuit where a known voltage is supplied.Sensitive detecting circuits are known, including voltage clampamplifiers and transimpedance amplifiers. The term “voltage clamp” hererefers to circuits which utilize a differential amplifier having oneinput connected to a variable command voltage, another input connectedto a measured voltage, and a feedback circuit. The voltage clamp usesnegative feedback to maintain the system at the command voltage, whichin this case is a predetermined alternating signal, such as analternating voltage signal from a signal generator. The output currentfollows changes in the input voltage and small changes in current can bedetected.

The term “quartz” means a nanopipette media is a fused silica oramorphous quartz, which is less expensive than crystalline quartz.Crystalline quartz may, however, be utilized. Ceramics and glassceramics and borosilicate glasses may also be utilized but accuracy isnot as good as quartz. The term “quartz” is intended and defined toencompass that special material as well as applicable ceramics, glassceramics or borosilicate glasses. It should be noted that various typesof glass or quartz may be used in the present nanopipette fabrication. Aprimary consideration is the ability of the material to be drawn to anarrow diameter opening. The preferred nanopipette material consistsessentially of silicon dioxide, as included in the form of various typesof glass and quartz. Fused quartz and fused silica are types of glasscontaining primarily silica in amorphous (non-crystalline) form.

The term “electrolyte” means a material that contains electrolytesolids, i.e., free ions. Typical ions include sodium, potassium,calcium, magnesium, chloride, phosphate and bicarbonate. Other ionicspecies may be used. The material will typically be liquid, in that itwill comprise the sample, containing the analyte, and the ions insolution. The sample itself may be an electrolyte, such as human plasmaor other body fluids, water samples and so on. The electrolyte shouldcarry an ionic current; about 10-100 mM, preferably about 100 mM ofpositive and negative ionic species are thought to be required for thisfunction. The present device may employ either the same or differentelectrolytes in the nanopipette interior and in the sample material.

The term “polyelectrolyte” is used herein in its conventional sense,i.e. polymers whose repeating units bear an electrolyte group. Thesegroups will dissociate in aqueous solutions (water), making the polymerscharged. Polyelectrolyte properties are thus similar to bothelectrolytes (salts) and polymers (high molecular weight compounds), andare sometimes called polysalts. Like salts, their solutions areelectrically conductive. Like polymers, their solutions are oftenviscous. Charged molecular chains, commonly present in soft mattersystems, play a fundamental role in determining structure, stability andthe interactions of various molecular assemblies. Polyelectrolytesinclude biological polymers which contain charged functional groups andsynthetic polymers. Examples of polyelectrolytes of biological origininclude, but are not limited to, oligonucleotides, nucleic acids,proteins, peptides, polysaccharides like pectin, carrageenan, alginates,and chitosan. Examples of synthetic polymers include, but are notlimited to, polyvinylpyrrolidone, carboxymethylcellulose, poly (sodiumstyrene sulfonated), polyacrylic acid, etc.

The charges on a polyelectrolyte may be derived directly from themonomer units or they may be introduced by chemical reactions on aprecursor polymer. For example, poly(diallyidimethylammonium chloride)(“PDAD”) is made by polymerizing diallyidimethylammonium chloride, apositively charged water soluble vinyl monomer. The positively-chargedcopolymer PDAD-co-PAC (i.e., poly(diallyidimethylammonium chloride) andpolyacrylamide copolymer) is made by the polymerization ofdiallyidimethylammonium chloride and acrylamide (a neutral monomer thatremains neutral in the polymer). Poly(styrenesulfonic acid) can be madeby the sulfonation of neutral polystyrene. Poly(styrenesulfonic acid)can also be made by polymerizing the negatively charged styrenesulfonate monomer.

Various polyelectrolytes comprising polyanions may be used in thepresent invention. Weak polyanions typically include carboxylic acidgroups while strong polyanions typically include sulfonic acid groups,phosphonic acid groups, or sulfate groups. Examples of anegatively-charged polyelectrolyte include polyelectrolytes comprising asulfonate group (—SO₃), such as poly(styrenesulfonic acid) (“PSS”),poly(2-acrylamido-2-methyl-l-propane sulfonic acid) (“PAMPS”),sulfonated poly(ether ether ketone) (“SPEEK”), sulfonated lignin,poly(ethylenesulfonic acid), poly(methacryloxyethylsulfonic acid), theirsalts, and copolymers thereof; polycarboxylates such as poly(acrylicacid) (“PAA”) and poly(methacrylic acid); and sulfates such ascarrageenin. Other polyanions include HV-sodium alginate, sodiumalginate, sodium hyaluronate, heparin sulfate, cellulose sulfate, kappacarrageenan, pentasodium tripolyphosphate, low-esterified pectin(polygalacturonic acid), polyglutamic acid, carboxymethylcellulose,chondroitin sulfate-6, chondroitin sulfate-4, and collagen. Themolecular weight and charge density of the polyanions are selected suchthat the compounds form polyelectrolyte complexes with a suitablepolycation.

Various polyelectrolytes, which are polycations, may also be employed ascationic polymers. Exemplary polycations include polyalkylene imines,such as polyethylene imine (“PEI”) and polypropylene imine. Otherpolycations include polyamines, i.e. polymers in which the monomer unitshave pendant amine groups, such as polyethylene polyamine, polypropylenepolyamine, polyvinylamine, polyallylamine,poly(vinylalcohol/vinylamine), chitosan, polylysine, polymyxin, sperminehydrochloride, protamine sulfate, poly(methylene-co-guanidine)hydrochloride, polyethylenimine-ethoxylated,polyethylenimine-epichlorhydrin modified, quartenized polyamide, andpolydiallyidimethyl ammonium chloride-co-acrylamide. As is known in theart, chitosan is a linear polysaccharide composed of randomlydistributed β-(1-4)-linked D-glucosamine (deacetylated unit) andN-acetyl-D-glucosamine (acetylated unit)

Other examples of a positively-charged polyelectrolytes includequaternary ammonium group, such as poly(diallyidimethylammoniumchloride) (“PDAD”), poly(vinylbenzyltrimethyl-ammonium) (“PVBTA”),ionenes, poly(acryloxyethyltrimethyl ammonium chloride),poly(methacryloxy(2-hydroxy)propyltrimethyl ammonium chloride), andcopolymers thereof; polyelectrolytes comprising a pyridinium group, suchas, poly(N-methylvinylpyridine) (“PMVP”), otherpoly(N-alkylvinylpyridines), and copolymers thereof; and protonatedpolyamines such as poly(allylaminehydrochloride) (“PAH”). The molecularweight and charge density of the polycations are selected such that thecompounds form polyelectrolyte complexes with a suitable polyanions.Further description of an pyridyl-based cationic polymer may be founde.g. in U.S. Pat. No. 4,384,075, “Cationic Alkenyl Azabenzenes andRubber Modified Asphalts.” Polyvinylpyrimidines such asPoly(4-vinylpyridine) (Mw ˜60,000), exemplified below, and, e.g.Poly(4-vinylpyridine-co-butyl methacrylate) are also explicitly definedas cationic polymers. Any of several groups can be used to alkylate thepyridyl-based polymer and produce a polycation. The groups can containreceptors such as boronic acid, biotin, and chelating ligands.Therefore, as is understood in the art, the present polyelectrolyte maybe a polyalkyl pyridine. i.e. a polymer having an alkyl backone withpendant pyridyl groups. A polyalkyl pyridine will typically have, asshown in FIG. 16, an alkyl (e.g. vinyl) backbone of “n” repeating units,depending on molecular weight (e.g. 1,000-10,000), and attached to thebackbone of certain, if not all monomer units, a pyridyl group eitherdirectly bonded to the monomer or bonded to the repeating monomer unitthrough a linker.

The term “salt” is used herein in its conventional sense, to refer toionic compounds that can result from the neutralization reaction of anacid and a base. They are composed of cations (positively charged ions)and anions (negative ions) so that the product is electrically neutral(without a net charge). These component ions can be inorganic such aschloride (Cr), as well as organic such as acetate (CH₃COO⁻) andmonatomic ions such as fluoride (F⁻), as well as polyatomic ions such assulfate (SO₄ ²⁻). There are several varieties of salts. Salts thathydrolyze to produce hydroxide ions when dissolved in water are basicsalts and salts that hydrolyze to produce hydronium ions in water areacid salts. Neutral salts are those that are neither acid nor basicsalts. Zwitterions contain an anionic center and a cationic center inthe same molecule but are not considered to be salts. Examples includeamino acids, many metabolites, peptides and proteins. Molten salts andsolutions containing dissolved salts (e.g. sodium chloride in water) arecalled electrolytes, as they are able to conduct electricity.

The term “polyacrylic acid” (PAA) means a polymer of acrylic acid units.The formula of PAA is (C₃H₄O₂)_(n). The number of repeating units may beselected to yield a polymer with molecular weight e.g. from 2,000 toabout 24,000. In a water solution at neutral pH, many of the side chainsof PAA lose their protons and acquire a negative charge. This makes PAAa polyelectrolyte, and a weak acid cation.

The term “polysaccharide” means polymeric carbohydrate structures,formed of repeating units (either mono- or di-saccharides) joinedtogether by glycosidic bonds. These structures are often linear, but maycontain various degrees of branching. Polysaccharides are often quiteheterogeneous, containing slight modifications of the repeating unit.Depending on the structure, these macromolecules can have distinctproperties from their monosaccharide building blocks. When all themonosaccharides in a polysaccharide are the same type the polysaccharideis called a homopolysaccharide, but when more than one type ofmonosaccharide is present they are called heteropolysaccharides.Polysaccharides have a general formula of C_(x)(H₂O)_(y) where x isusually a large number between 200 and 2500. Examples include, but arenot limited to storage polysaccharides such as starch and glycogen;structural polysaccharides such as cellulose, chitin and arabinoxylans;bacterial polysaccharides like peptidoglycan. Other examples includepectin, carrageenan, alginates, chitosan, etc.

Ion binding polysaccharides are those such as the exemplified chitosan,xanthan, alginic acid, chitin, and pectin. An ion binding polysaccharideas described here works by chelation.

The term “chelation” is used in its conventional sense, to refer to theactivity of a chelating agent. A chelate is a chemical compound composedof a metal ion and a chelating agent. A chelating agent is a substancewhose molecules can form several bonds to a single metal ion. In otherwords, a chelating agent is a multidentate ligand. Chelants, accordingto ASTM-A-380, are “chemicals that form soluble, complex molecules withcertain metal ions, inactivating the ions so that they cannot normallyreact with other elements or ions to produce precipitates or scale.”

The term “chitosan” is used herein in its conventional sense, to referto is a linear polysaccharide composed of randomly distributedβ-(1-4)-linked D-glucosamine (deacetylated unit) andN-acetyl-D-glucosamine (acetylated unit). The amino group in chitosanhas a pKa value of ˜6.5, which leads to a protonation in acidic toneutral solution with a charge density dependent on pH and the %DD(degree of deacetylation) value. This makes chitosan water soluble and abioadhesive which readily binds to negatively charged surfaces such asmucosal membranes. Chitosan enhances the transport of polar drugs acrossepithelial surfaces, and is biocompatible and biodegradable. Chitosan isproduced commercially by deacetylation of chitin, which is thestructural element in the exoskeleton of crustaceans (crabs, shrimp,etc.) and cell walls of fungi. The degree of deacetylation (%DD) can bedetermined by NMR spectroscopy, and the %DD in commercial chitosans isin the range 60-100%. On average, the molecular weight of commerciallyproduced chitosan is between 3800 to 20,000 daltons. Chitosan has beendescribed as a suitable biopolymer for the collection of metal ionssince the amino groups and hydroxyl groups on the chitosan chain can actas chelation sites for metal ions. Further details on copper binding bychitosan may be found in Food Chemistry 114 (2009) 962-969. Chitosan hasbeen described as a suitable biopolymer for the collection of metal ionssince the amino groups and hydroxyl groups on the chitosan chain can actas chelation sites for metal ions. It has a structure illustrated, e.g.in US 2011/0136255.

The term “calmodulin” (an abbreviation for CALcium MODULated proteIN) isused herein in its conventional sense, refers to a calcium-bindingprotein expressed in all eukaryotic cells. It can bind to and regulate anumber of different protein targets, thereby affecting many differentcellular functions. Calmodulin undergoes a conformational change uponbinding to calcium, which enables it to bind to specific proteins for aspecific response. Calmodulin can bind up to four calcium ions, and canundergo post-translational modifications, such as phosphorylation,acetylation, methylation and proteolytic cleavage, each of which canpotentially modulate its actions. Calmodulin is a small, acidic proteinapproximately 148 amino acids long (16706 Daltons) and, as such, is afavorite for testing protein simulation software. It contains fourEF-hand “motifs”, each of which binds a Ca²⁺ ion. The protein has twoapproximately symmetrical domains, separated by a flexible “hinge”region. Calcium is bound via the use of the EF hand motif, whichsupplies an electronegative environment for ion coordination. Aftercalcium binding, hydrophobic methyl groups from methionine residuesbecome exposed on the protein via conformational change. This presentshydrophobic surfaces, which can in turn bind to Basic AmphiphilicHelices (BAA helices) on the target protein. These helices containcomplementary hydrophobic regions.

Calmodulin is an exemplified ion binding protein that binds calcium.Other calcium binding proteins include troponin C and S100B. Otherproteins that are not naturally metal ion binding proteins can becoupled to small molecule chelating agents. Other suitable ion bindingproteins include CopA and metallothionein (binding copper), zinc fingerproteins, cytidine deaminase, and nerve growth factor (binding zinc).

The term “boronic acid” is used herein in its conventional sense, torefer to an alkyl or aryl substituted boric acid containing acarbon-boron bond belonging to the larger class of organoboranes.Boronic acids act as Lewis acids. Their unique feature is that they arecapable of forming reversible covalent complexes with sugars, aminoacids, hydroxamic acids, etc. (molecules with vicinal, (1, 2) oroccasionally (1, 3) substituted Lewis base donors (alcohol, amine,carboxylate)). The pKa of a boronic acid is ˜9, but upon complexion inaqueous solutions, they form tetrahedral boronate complexes with pKa ˜7.They are occasionally used in the area of molecular recognition to bindto saccharides for fluorescent detection or selective transport ofsaccharides across membranes. Boronate esters are esters formed betweena boronic acid and an alcohol. Boronic acids have the formula RB(OH)₂,where R can be any group, e.g. alkyl. A boronate ester has the formulaRB(OR)₂.

The covalent pair-wise interaction between boronic acids and 1,2- or1,3-diols in aqueous systems is rapid and reversible. As such theequilibrium established between boronic acids and the hydroxyl groupspresent on saccharides can be employed to develop a range of sensors forsaccharides. Potential applications for this interaction include systemsto monitor diabetic blood glucose levels.

Boronic acids and boronic esters may be used to functionalize thenanopipettes embodied herein to be used in detecting saccharidesincluding monosaccharides, disaccharides, oligosaccharides andpolysaccharides, including glucose, a monosaccharide. Monosaccharidesare the simplest carbohydrates in that they cannot be hydrolyzed tosmaller carbohydrates. They are aldehydes or ketones with two or morehydroxyl groups. The general chemical formula of an unmodifiedmonosaccharide is (C.H₂O)_(n), literally a “carbon hydrate.”Monosaccharides are important fuel molecules as well as building blocksfor nucleic acids. The smallest monosaccharides, for which n=3, aredihydroxyacetone and D- and L-glyceraldehyde. Two joined monosaccharidesare called a disaccharide and these are the simplest polysaccharides.Examples of disaccharides that could be sensed with a boronic acidcoating include sucrose and lactose. Examples of analytes that areoligosaccharides include disaccharides, the trisaccharide raffinose andthe tetrasaccharide stachyose. Examples of polysacchrides includestarch, glycogen, chitin, cellulose, callose or laminarin,chrysolaminarin, xylan, arabinoxylan, mannan, fucoidan andgalactomannan.

The term “saccharide binding protein” means a protein that specificallybinds to a carbohydrate. The term “saccharides” is used synonymouslywith “carbohydrates”. An example of carbohydrate binding proteins is thefamily of lectins. Examples include concanavalin, mannose-bindingprotein, peanut agglutinin, snowdrop lectin, ricin.

General Method and Apparatus

Described herein is a method and apparatus to control precipitation inaqueous solutions by voltage-directed ion migration, and to study theprecipitation through its effect on ion currents through a nanopipettetip. Studying the earliest stage of precipitation at the nanoscale istechnically challenging, but quite valuable as such phenomena reflectimportant processes such as crystallization and biomineralization. Usinga quartz nanopipette as a nanoreactor, precipitation of an insolublesalt is induced to generate oscillating current blockades. Thereversible process can be used to measure both kinetics of precipitationand relative size of the resulting nanoparticles.

FIG. 1 shows an example of an electrochemical setup according to thepresent invention measuring ion current oscillations through a quartznanopipette 102, which has its tip in a solution 106 containing variousionic and/or carbohydrate species 112. Electronics are provided formeasurement of ion current oscillations in the nanopipette. Ioniccurrents result from the flow of ions into or out of the nanopipettethough the nanopore in response to a voltage differential between theinside and the outside of the nanopipette. The electronics include anelectrode 104 in contact with the solution within the pipette; anamplifier 105 such as an Axopatch resistive feedback patch clamp andhigh speed current clamp amplifier, manufactured by Molecular Devices,with the negative input connected to the electrode 104; and a referenceelectrode 110 in the sample solution 106, outside the interior of thenanopipette. This exemplary setup measures ion current through a quartznanopipette. Pore diameter at the tip is typically 40-60 nm. Asexemplified below and shown here, solutions contain various ions such asKCl (0.1 M) and are buffered at pH 7, with 10 mM potassium phosphate inthe barrel 102 and 10 mM Tris-HCl in the bath 104. Zinc chloride isincluded in the bath at concentrations of 2 to 20 μM. The use of aphosphate-free buffer in the bath is to prevent precipitation in thebath solution. As such, the precipitation is localized to the tip of thenanopipette when phosphate from inside the barrel mixes with zinc ionsfrom the bath solution. Further details regarding the voltage clampcircuit may be found in U.S Pat. No. 7,785,785, “Charge perturbationdetection system for DNA and other molecules.”

The same device in a different configuration can be used to measure ioncurrent rectification as a means to detect metal ions, as illustratede.g. in FIGS. 4 and 9. In this embodiment, a buffer such as pH 7phosphate is as the electrolyte for both the barrel of the nanopipetteand the bath solution. Also shown in FIG. 1 is a coating 108 on theinside bore, adjacent the nanopipette tip. This region is referred to asa “nanopore,” and defines within it a “nanochannel.” The coating 108 asfurther described below may extend in an a mesh-like structure acrossthe tip, or may be a single coating on the interior surface, and may beused for specific binding of selected ions or carbohydrates. Asdescribed below, polyacrylic acid may be applied as shown at 108, i.e,applied to the silicate surface of the nanopipette and chitosan isapplied on top of the polyacrylic acid. Cu ions are shown binding to thechitosan, but being released at pH 3.

Sensing metal ions by using a chelator differs from measuring changes incurrent oscillations due to nanopreciptiation at the nanopore. When thechelator binds catins, negative ionic current rectification indicatessuch binding, as described below.

The polymer is synthesized such that it is cationic, and the polymer,when embedded in the nanopore, displays positive current rectification.On binding saccharides, the positive charge is neutralized, and the ioncurrent becomes negatively rectified. This is not the case for othersystems in the literature in which boronic acid is attached directly tothe glass. Those systems achieve modest changes in rectification(negative to slightly more negative) on sugar binding.

As is known, an I-V curve (current voltage curve) will exhibit variouscharacteristics depending on the flow of charged ions through ananopore. Where rectification takes place, the IV curve will not belinear, but will greater passage of current in response to a positivevoltage (positive rectification) or in response to a negative voltage(negative rectification). The polarity of the voltage is given withreference to the electrode within the nanopipette. Thus, in operation,the present device will apply a number of voltage levels successively inthe negative range, then in the positive range. For example, in FIG. 4,voltages of −400 mV to 0 and from 0 to +400mV were applied in 14discrete levels and currents measured at each level, in order togenerate an I-V curve. This range may be narrowed when specificparameters of a given system are determined.

FIG. 2 shows a configuration of a nanopipette circuitry and solutionscausing ion current oscillations. A negative potential in thenanopipette barrel 202 (shown as V<0) draws zinc cations from the bath106 (FIG. 1) into the pore, i.e. the opening of the nanopipette, whilephosphate ions are pushed out of the nanopipette barrel 102 (FIG. 1).Mixing of the ions (anion mixing with cation) occurs in the nanopipettetip region 118. When a precipitate of sufficient size is formed, asshown at 206, the pore is blocked and ionic current decreases.

As described below, counter ions for the highly water insoluble saltzinc phosphate were separated by the pore of a nanopipette and apotential applied to cause ion migration to the interface. By analyzingthe kinetic of the pore blockage, illustrated by a mass at 206, twodistinct mechanisms were identified: a slower process due toprecipitation from solution, and a faster process attributed tovoltage-driven migration of a trapped precipitate. These techniques canbe used to study precipitation dynamics and carry out measurement ontrapped particles within a nanoreactor, which may be considered to bethe “reaction zone” as shown in FIG. 2. In the example given, theparticles are zinc phosphate salts. Other particles can includeproteins, especially those that are charged, and the nanopipette can beused to test conditions for seeding crystals such as those used incrystallography. The device can also be used as an electrical sensor foreither cations or anions based on nanoprecipitation with counter ions.

Also disclosed is an electrical sensor that reversibly binds ions usinga nanopipette functionalized with receptors. Examples given are sensorsfor pH, calcium, copper, and carbohydrates. The receptor can bemolecules with acidic or basic functionalities, metal chelators, orproteins. As a proof of concept for a calcium biosensor, the proteincalmodulin was immobilized to the interior of a nanopipette tip. Thesensor showed selectivity for calcium over magnesium in electrolytes ofneutral pH, and the calcium signaling was reversible simply by immersingin fresh solution. This sensor was used for over 20 separatemeasurements with reproducible and concentration-dependent signals.

Furthermore, it was demonstrated that a nanopipette functionalized withcertain polyelectrolytes reversibly bind transition metals. As a proofof concept, nanopipettes modified with chitosan as an external layer andpolyacrylic acids (PAA) layered onto the quartz interior were shown toreversibly bind copper. In this case, chitosan/PAA is applied, asdescribed below. The outside of the nanopipette is treated, e.g. withsilane. The Cu ions bind to chitosan that is combined with thepolyacrylic acid on the inner surface of the nanopipette. At pH 3, theCu 2+ ions come off of the chitosan. The surface treatment of theoutside of the nanopipette may be carried out to facilitate penetrationof the nanopipette through membranes, for example, lipid bilayers, cellmembranes. The surface treatment of the outside of the nanopipette doesnot alter the binding of the receptors to the nanopipette.

Moreover, as shown below, the applied voltage can be employed to tunethe binding properties of the nanopipette. In some embodiments, thenanopipette is functionalized with proteins to bind to ions, includingto carbohydrates. In some other embodiments, the nanopipette isfunctionalized with boronic acids or boronic esters to detectcarbohydrates. Additionally, sensors for pH were prepared byfunctionalizing the nanopipette with either polyelectrolytes containingamines or carboxylic acid groups, the amine-containing biopolymerchitosan, or an amino-silane. The pH sensors also showed rapidlyreversible response to different buffers from pH 3 to pH 8. These typesof sensors may be used for many different applications requiring areversible and continuous sensor or array of sensors, such as monitoringof water quality, in-vivo single-cell assays, or functional ion mapping.

EXAMPLES Example 1 Preparation and Characterization of NanopipetteBiosensors with PLL and PAA Polyelectrolyte Layers

Quartz capillaries with filament (QF100-70-7.5) from Sutter (Novato,Calif.) were used as received and pulled with a Sutter P-2000 laserpuller to give nanopipettes. Puller settings used were heat 620,filament 4, velocity 60, delay 170, pull 180. The settings are variabledepending on the puller, and were adjusted as needed to providenanopipettes showing negative ion current rectification with the desiredconductance. The pipettes were backfilled with a buffered electrolyte(pH 7 Tris-HCl, 10 mM and KCI, 100 mM) unless otherwise indicated. Thetwo sensors described are twin pipettes from one pulled capillary.Sensor CaM-1 was untreated prior to polyelectrolyte deposition, andCaM-2 was silanized with trimethylchlorosilane (TMCS) using vapordeposition. The nanopipette was placed in a sealed chamber of 0.5 Lvolume with approximately 0.1 mL of TMCS for 10 minutes. Both pipetteswere then backfilled with buffered electrolyte and immersed in a bath ofthe same buffer. The ion current was measured with an Axopatch 700Bamplifier (Axon) using Ag/AgCl electrode in the pipette barrel and aground electrode in the bath. A sinusoidal potential from +500 mV to−500 mV (5 Hz) was applied to monitor the ion current during subsequentsurface treatment. The polyelectrolytes poly-L-Iysine PLL) andpolyacrylic acid (PAA) were deposited on the surface of the nanopipetteby sequential immersion of the pipette tip into buffered electrolytecontaining either PLL or PAA at a concentration of 3 ppm, with immersionin buffer to wash after each polyelectrolyte deposition. Apolyelectrolyte layer was determined to be stable if the resultingchange in current rectification (positive for PLL, negative for PAA) wasmaintained during immersion in buffer. Both CaM-1 and CaM-2 werefunctionalized with four layers: PLL, PAA, PLL, and then PAA. Thepipettes were then immersed in a solution containing 10 mg/mL each ofNHS and EDC (100 mM pH 6.1 MES buffer, with 50 mM KCl) for one hour.Finally, the tips of the pipettes were washed and immersed in a solutionof calmodulin (bovine brain, 0.05 mg/mL in pH 6.1 MES buffer (100 mM)with 50 mM KCl) and incubated for 18 h at 4° C.

The electrical properties of the sensors and response to metal saltswere analyzed using the electrical setup described above. Allmeasurements were carried out in pH 7-buffered electrolyte solution withaliquots of either calcium chloride or magnesium chloride (1 to 10 μLvolumes) directly added to a bath of 0.3 mL buffer. Data was sampled ata rate of 200 Hz using the pClamp software and was processed usingOriginPro 8.5. For continuous measurement data, the negative ion currentpeaks arising from the sinusoidal applied voltage were detected andplotted as a function of time. Line smoothing was done with a 50%percentile filter and a 10-point moving window, Current rectificationcoefficient (r) was calculated using the following equation: r=log₁₀I₊/I⁻

where I₊ is the magnitude of the ion current at a potential of 500 mV,and I⁻ is the magnitude of the ion current at a potential of −500 mV.Errors in ion current reflect the standard deviation between threeseparate measurements of I₊ and I⁻, with the same nanopipette afterwashing in buffer between measurements.

Example 2 Selective and Reversible Ca2+ Binding by CalmodulinImmobilized to a Nanopipette

To achieve reversible and selective ion binding with a biologicalreceptor, nanopipette sensors modified with calmodulin, a calciumbinding protein that reversibly chelates calcium (K_(d) ˜10⁻⁶ M) withhigh selectivity, were studied. Electrical sensors using immobilizedcalmodulin have been previously reported for probing both calciumconcentration by Cui et al. in Science vol. 293: pages 1289-1292 in 2001(“Nanowire nanosensors for highly sensitive and selective detection ofbiological and chemical species”) and protein-protein interactions byIvnitiski et (“An amperometric biosensor for real-time analysis ofmolecular recognition” Bioelectrochem. Bioenerg. 1998, 45(1), 27-32) andby Lin et al. (“Label-free detection of protein-protein interactionsusing a calmodulin-modified nanowire transistor” Proc. Natl. Acad. SetU.S.A. 2010, 107(3), 1047-1052). Our strategy took into considerationboth immobilization of the protein to the pore, as well as the need tolocalize the receptor to the pore solution interface for rapid andreversible ion response.

In preliminary studies, the highest sensitivity to cations was observedwith pores showing negative rectification, and our approach to surfacefunctionalization ensured that the final sensor is based on such pores.Polyelectrolytes have strong electrostatic interactions with the chargedpore surface, and the ion current rectification is an excellentindicator of polyelectrolyte binding to the nanopore. With this in mind,ion current measurements were used to monitor the layer-by-layerdeposition of polyelectrolytes in the nanopipette and provide anegatively rectified nanopore with desired conductance. Amide bondformation between the amine groups in the protein and carboxylate groupson the outermost polyelectrolyte layer coupled the protein to the quartzsurface. To localize the receptor to the pore-solution interface wherethere is greater interaction with the bulk solution, the pore wasfunctionalized only by immersion, leaving the inside of the nanopipettefilled with buffer. Because such an approach will functionalize arelatively large area of the surface in addition to the region directlyaround the pore, nanopipettes in which the external surface was coatedwith a hydrophobic silane was also tested.

The surface-treatment of the nanopipettes was monitored in real-time todetermine stability of the surface chemistry for several steps. Twinnanopipettes, pulled from the same capillary, were taken throughidentical steps of surface treatment. The treatment involved reactionwith pendant oxygen groups on the surface of the silica. PAA and PLLlayers were applied, resulting in pendant carboxyl groups; calmodulinwas bound to these groups. Thus surface functionalization on quartz wasaccomplished by deposition of polyelectrolyte layers (poly-L-lysine)PLL; (poly-acrylic acid) PAA, followed by amide bond formation to CaM(calmodulin) protein using NHS/EDC coupling. In effect, there is createda sandwich of quartz—PLL-PAA-PLL-PAA.

Sensor CaM-1 was used directly after pulling, and CaM-2 was firsttreated with trimethylchlorosilane (TMCS) vapor to silanize the outerpipette tip. The ion current rectification (ICR) was monitored as thenanopipettes were immersed in neutral buffer containing either cationicpoly-L-lysine (PLL) or anionic polyacrylic acid (PAA), with addition ofsubsequent layers only after the rectification remained stable in purebuffer. As shown by a current-voltage plot in FIG. 4, the barenanopipette has a negative ICR, where current-voltage response of thebare pipette (▪), after two layers of PLL and PAA (●), and aftercoupling with CaM (▴). After two layers of PLL/PAA, the current is stillnegatively rectified, but smaller in magnitude indicating a smaller poreafter deposition. This behavior continues after immobilization ofcalmodulin protein, which is also negatively charged at neutral pH(pI˜4). The rectification coefficient reflects the behavior seen in thecurrent voltage curves: r=−0.27±0.03 (bare pipette), −0.71±0.02 (secondlayer of PLL/PAA), and −0.533±0.014 (CaM). The low error in thesemeasurements demonstrates the stability of the surface at each step. Thecurrent rectification with the nanopipettes reflected the surfacecoating (see FIG. 5A, showing rectification coefficients that differwith different functionalizations). After applying PLL, the currentrectification had a positive value, while after applying PAA, therectification was negative. The current was measured with an oscillatingsinusoidal potential (−500 to 500 mV, 5 Hz). Error bars reflect threeseparate measurements with the same nanopipette, with washing in bufferbetween each measurement. The CaM-modified nanopipette also showednegative current rectification, consistent with the overall negativecharge of the protein resulting from carboxylate-containing residues.The current at a potential of −500 mV is strongly affected by thepresence of calcium ions, as shown in FIG. 5B. The ion current is shownas a function of time in pH 7 buffer, with addition of 0.1 mM magnesiumchloride, and in the presence of 0.1 mM calcium chloride. Theselectivity for calcium is illustrated by the larger signal change forcalcium ions relative to magnesium ions. The reversibility of thebinding is shown by restoration of the signal by immersing in purebuffer, followed by calcium chloride.

Example 3 pH-Sensitive Nanopipette Sensors Functionalized with Metal IonBinding Polymer (Chitosan Binding Cu)

Sensors of pH were prepared by functionalizing the nanopipette witheither an amino-silane, the amine-containing biopolymer chitosan (FIG.6) or polyelectrolytes containing amines or carboxylic acid groups. ThepH sensors showed rapidly reversible response to different buffers frompH 3 to pH 8. The nanopipette was filled with pH 7 electrolyte (100 mMKCl with 10 mM Tris-HCl buffer) and dipped in buffered electrolytes ofvarying pH (100 mM KCl with 10 mM phosphate/citrate buffer). Ion currentwas measured while applying a sinusoidal potential from −500 to 500 mVat 5 Hz. Measurements were carried out in a 0.1 M KCl solution, bufferedwith 10 mM phosphate/citrate to the desired pH. Error bars werecalculated from at least four different pH measurements with the samesensor (FIG. 6). FIG. 6 shows a comparison of the rectificationcoefficient for bare and chitosan/PAA-functionalized nanopipettes atdifferent pH values. The rectification coefficient is more sensitive topH for the functionalized nanopipette, owing to the protonablecarboxylate and amine groups. At neutral pH, the functionalizednanopipette shows significantly more negative rectification than thebare nanopipette, showing that the coating results in more negativelycharged groups at the surface of the nanopore.

Example 4 Electrostatic Physisorption of Polyelectrolytes and Chitosanon a Nanopipette

Chitosan and polyacrylic acid were physisorbed using the followingprocedure: Nanopipette was immersed into 350 μL of a pH 3 bufferedsolution and 10 μL of the chitosan stock solution were added in thereservoir. The physisorption of chitosan should take place at acidic pHsince this polyelectrolyte is not soluble in neutral pH. The nanopipettewas then transferred into a 350 μL of a pH 7 buffered solution and 10 μLof the PAA stock solution was added in the reservoir. The functionalizednanopipettes were then cycled between the two solutions until thedesired number of layers on the sensor was reached.

The conical geometry as well as the nanometer size pore generates aninteresting electrochemical behavior on solid state nanopore. Forinstance, charged nanopores respond to a symmetric input voltage with anasymmetric current output, an effect referred to as currentrectification. The origin of this effect in nanopipettes has beenextensively described in a recent review published by two of the presentinventors (Actis, P.; Mak, A.; Pourmand, N. Bioanalytical Reviews 2010,1, 177.). Briefly, when the diffuse electrical double layer thickness iscomparable with the pore size, the electrostatic interactions betweenfixed charged species on the nanopore surface and ionic species insolutions alters ion transport properties. In order to quantify theextent of the rectification, it has been introduced an useful parameterdenoted as rectification coefficient or, in some cases, degree ofrectification that is defined as the logarithm of the ratio between thecurrent measured at particular positive voltage and the current measuredat the same voltage but with the opposed polarity,. i.e. r=Log₁₀ I+/I−.

Quartz nanopores, being negatively charged, show a negative currentrectification (r<0). The rectification can be inverted (r>0) bymodifying the nanopore surface with charged functional layers such aspoly-1-lysine, dendrimers, aminosilane, and chitosan.

The electrostatic physisorption of polyelectrolytcs can be monitored bysimple electrochemical measurements. The positively charged amino groupsfrom the chitosan backbone allow the physisorption of thepolyelectrolyte on the negatively charged nanopipette surface. Chitosanphysisorption occurs at acidic pH only since this polysaccharide isinsoluble at neutral pH. Similarly, carboxylic groups of polyacrylicacid (PAA) confer to the polymer a negative charge at neutral pHallowing the physisorption on the positively charged chitosannanopipette. The deposition of every polyelectrolyte layer was monitoredby electrochemical measurement. The rectification coefficient is anindication of the nanopipette surface charge. This parameter wasemployed to quantify the effect of the layer-by layer assembly on thequartz nanopipette. Interestingly, the multilayer assembly increased therectification properties of the nanopipette: the rectificationcoefficient at pill increased from −0.1, for a bare nanopipette, to−0.8, after the physisorption of 5 layers of chitosan/PAA and plateauedafterwards. Similarly at pH 3 the rectification coefficient increasedfrom 0 to 0.65 after 5 layers (FIG. 7). Besides the rectificationcoefficient, after 5 layers, there was no change in the overall currentupon further additions of PAA or chitosan. This indicates that nopolyelectrolyte was deposited on the sensor surface that was alreadyfully covered with a chitosan PAA mixed layer. These results contrastwith the ones described by Ali et al. (Ali, M.; Yameen, B.; Cervera, J.;Ramirez, P.; Neumann, R.; Ensinger, W.; Knoll, W.; Azzaroni, 0. Journalof the American Chemical Society 2010, 132, 8338) that showed that thesurface charge of a single asymmetric nanochannel in a PET membranedecreases dramatically with the number of layers assembled into it. Thisbehavior can be explained by the imperfect multilayer formation that ledto a mixed layer rather than a perfect layer by layer assembly.Therefore, the pH response of the chitosan/PAA modified nanopipette wasstudied to corroborate this assumption. Measurements were carried out ina 0.1 M KCl solution, buffered with 10 mM phosphate/citrate to thedesired pH. Error bars were calculated from at least four different pHmeasurements with the same sensor (FIG. 8). Chitosan has a pK_(a) valueof ˜6.5, while PAA of 4.8. Assuming a perfect layer-by layer assembly,if PAA is the outermost layer, the nanopipette should be neutral atpH<4.8 and negatively charged at pH>4.8. Likewise, if chitosan is theoutermost layer, the nanopipette should be positively charged at pH<6.5and neutral charged above it. The rectification coefficient of thechitosan/PAA nanopipette, however, is positive at pH<5, and negative atpH>5. This indicates that at pH<5, the nanopipette permselectivity isgoverned by the protonated amino groups of chitosan while, at pH>5, bythe negatively charged carboxylic groups of the PAA, thus demonstratingthe mixed layer formation.

Example 5 Selective and Reversible Cu2+ Binding on Quartz NanopipettesElectrostatically Modified with Chitosan and PAA Multilayers

The physisorption of chitosan and PAA layers on a quartz nanopipettegives it reversible metal binding properties that are not observed withthe bare sensor. Chelating properties of chitosan and PAA are well knownand well described in the literature. Chitosan binds several metal ions;however it shows a stronger affinity for cupric ions. Thus it wasdecided, as a model system, to study the complexation of copper ions tochitosan/PAA sensors.

Reagents: Chitosan was purchased from CERMAY. A stock solution of 5mg/ml chitosan into pH 3 HCl solution was prepared and used for all theexperiments described in this paper. Polyacrylic acid was purchased fromSigma Aldrich (Saint Louis, Mo.). PBS solutions at pH 7.4 were preparedusing standard method. Aqueous reagents were prepared using ultrapurewater with>18 MΩ cm⁻¹ resistance.

Sensor fabrication: Nanopipettes were fabricated from quartz capillarieswith filaments, with an outer diameter of 1.0 mm and an inner diameterof 0.70 mm (QF100-70-5; Sutter Instrument Co.). The capillary was thenpulled using a P-2000 laser puller (Sutter Instrument Co.) preprogrammedto fabricate nanopipettes with an inner diameter of 50 nm. Parametersused were: Heat 625, Fil 4, Vel 60, Del 150, and Pul 192. The resultingnanopipette tips had inner diameters ranging from 37 to 82 nn, with themean diameter of 56 nm.

Measurement Setup: All the measurements were performed in a twoelectrode setup since the current flowing through the nanopipette is toosmall to polarize a reference electrode. The sensor, acting as theworking electrode, is backfilled with a 0.1 M KCl, 10mM Tris-HClbuffered at pH7, and a Ag/AgCl electrode is inserted. Another Ag/AgClground electrode is placed in bulk solution acting asauxiliary/reference electrode. Both electrodes are connected to theAxopatch 700B amplifier with the DigiData 1322A digitizer (MolecularDevices), and a PC equipped with pClamp 10 software (Molecular Devices).The system remained unstirred for the duration of the measurement, whichwas conducted at room temperature.

FIG. 9 shows the ion current at potentials of −500 and +500 mV for abare nanopipette sensor and one that has been coated with PLL/PAA. Theaddition of Cu2+ in the reservoir immediately affects thepermselectivity of the sensor causing a decrease in the ionic current ata potential of −500 mV. The binding is completely reversible and sensorsare regenerated up to 5 times without any loss of performance.Regeneration is performed by immersion of the sensor into a pH3 bufferfor 60 seconds. Acidic pH protonates chitosan amino groups thus causingthe release of the cupric ions in solution (FIG. 10). Alternativemethods of regeneration such as immersion into citrate buffer at neutralpH and 0.1% EDTA showed equal success. It is important to consider thecombined effect of both polyelectrolytes for the copper binding.Experiments were performed when the nanopipette was functionalized withchitosan and PAA only that showed little variation in the output currentupon addition of copper in the bulk solutions. Furthermore, theinterfaces between the polyelectrolytes and the quartz were not stableas the sensors were regenerated only once before a complete loss of thecopper binding property. When mixed layers of chitosan and PAA wereconstructed on a nanopipette, the interface was stable over multipledetection cycles. Through FTIR measurements, Wang and coworkersdemonstrated that —NH2, —OH and COOH groups were all involved in thecopper adsorption by chitosan/PAA attapulgite composites (Wang, X.;Zheng, V.; Wang, A. Journal of Hazardous Materials 2009, 168: 970). Itwas speculated that a similar chelation mechanism takes place in thechitosan/PAA sensor enhancing the metal binding ability.

The response of the sensor to different concentrations of copper ionswas the investigated (FIG. 11). The sensor responds linearly toincreasing Cu²⁺ concentrations (FIG. 11, inset).The variation of thenormalized current vs. Cu²⁺ concentration is analogous to a Langmuiradsorption isotherm. The current was normalized according to:

$I_{\mathfrak{n}} = {1 - \frac{I_{s}}{f_{b}}}$

Where I_(s) is the signal after addition of copper ions in solution, andIb is the baseline signal measured in pure buffer. Assuming that thebinding process is an equilibrium process, the variation of thenormalized current is proportional to the number of cupric ions bound tothe sensor, the binding sites are independent, and that the complexationequation is given by:

Cu²⁺(aq)+Chitosan/PAA↔[Cu²⁺−Chitosan/PAA]

One can estimate the thermodynamic affinity constant K for Cu²⁺ bindingto the sensor using the following equation:

$\frac{1}{I_{n}} = {\frac{1}{I_{\max}} + \frac{1}{I_{\max}{Kc}_{{cu}^{2 +}}}}$

Where I_(max) is the I_(n) value at maximal surface coverage, and c isthe concentration of cupric ions in solution. From the linear fit ofFIG. 11, a K value of 4×10⁴ M⁻¹ can be extrapolated. This value is ingood agreement with the ones calculated for cation adsorption tochitosan with different platforms.

Example 6 Influence of the Waveform, Amplitude and Frequency of theApplied Voltage

The applied voltage, i.e. the voltage between an electrode containingthe solution inside of the nanopipette and an electrode in the externalsolution, plays a crucial role in the detection mechanism. Molecules canbe trapped or concentrated at the tip of a nanopipette. Furthermore, theapplied voltage increases the probability of a binding event inside thesensing region of the sensor. First, how the amplitude and the frequencyof a sinusoidal waveform affect the nanopipette electrical single uponbinding of copper ions was studied Response of the sensor to a fixedconcentration of Cu2+ (20 uM) as a function of the (a) amplitude and (b)frequency of the applied voltage was measured in a bath solution: 0.1MKCl, 10 mM Tris-HCl, pH 7.

A higher voltage applied gave a larger change in the output current uponchelation of cupric ions by the chitosan/PAA sensor. For an equal copperconcentration (20 μM), the current decreased to 5% of its initial valueat 1 V amplitude while only 46% decrease was detected with 50 mVapplied.

Interestingly, the higher the frequency of the applied sinusoidalvoltage the smaller was the change measured upon binding of copper bythe sensor. For a 20 μM Cu²⁺ concentration in the bulk solution, thecurrent decreased to 68% of its initial value at 1 KHz frequency whileto 58% at 0.5 Hz.

Once characterized the response of the sensor to an AC voltage, theeffect of a DC voltage was investigated. The binding of copper on thesensor can be controlled by the applied voltage. When a positive voltageis applied, cations are depleted from the nanopipette tip due to theelectrophoretic flow. Leveraging this effect the binding of copper onthe sensor can be triggered by controlling the applied voltage. Upon apositively applied voltage no binding occurs as cupric ions are depletedfrom the nanopipette tip, as soon as the voltage is switched tonegative, binding occurs causing a decrease in the ion flow, a changethat is reflected on the next positive step (FIG. 13).

Example 7 Voltage-Gated Nanoreactor for Detection of Precipitation withNanopipette

This example discloses how a voltage bias across a nanopipette openingcan be employed to control ion migration and cause precipitation of aninsoluble salt at the interface of two aqueous media. Furthermore, theconditions required for generating oscillating current due to zincphosphate precipitation in a nanopore, as well as investigations intothe nature of the precipitate and its subsequent evacuation from thepore are described. Further it is shown that a pore which appearspermanently blocked by precipitation can be briefly cleared with avoltage pulse, and the method is used to examine the kinetics of poreblockage.

Reagents and Solutions

Stock solutions of metal salts (100 to 500 mM) were prepared in Milli-Qultrapure water with 5% HCl. These were then diluted in buffer the dayof the experiment. Calcium chloride tetrahydrate was purchased fromFisher. Zinc chloride, iron(III) chloride, and magnesium chloride (1.00M solution) were purchased from Sigma-Aldrich. Buffer solutions wereprepared from potassium chloride (Baker), sodium phosphate, dibasic(Sigma), and TRIS-HCl (1 M solution, pH 7.00, Sigma) and adjusted witheither HCl (1 M) or KOH (0.1 M). All buffer solutions used for analysiscontained 10 mM buffer and 100 mM potassium chloride.

Quartz Nanopipette Fabrication

Nanopipettes were fabricated from quartz capillaries with filaments,with an outer diameter of 1.0 mm and an inner diameter of 0.70mm(QF100-70-5; Suffer Instrument Co.). The capillary was then pulled usinga P-2000 laser puller (Suffer Instrument Co.) preprogrammed to fabricatenanopipettes with an inner diameter of approximately 50 nm. Parametersused were: Heat 625, Filament 4, Velocity 60, Delay 170, and Pull 180.In a solution of 10 mM buffer and 100 mM KCl, the pipettes gave acurrent between −2500 and −4000 pA at a potential of −0.5 V.

Measurement Setup

For measuring ionic current through a nanopipette, a two electrode setupwas used. The nanopipette was backfilled with buffer solution and anAg/AgCl electrode inserted. Another Ag/AgCl electrode was placed in 0.3mL bulk solution acting as auxiliary/reference electrode. Bothelectrodes were connected to an Axopatch 700B amplifier with theDigiData 1322A digitizer (Molecular Devices), and a PC equipped withpClamp 10 software (Molecular Devices). Positive potential refers toanodic potential applied to the electrode in the barrel of the pipetterelative to the counter electrode. Experiments were carried out at 24°C.

Voltage-Driven Nanoprecipitation

To induce zinc phosphate precipitation by voltage-driven mixing, thebarrel of a nanopipette was backfilled with a solution of phosphatebuffered electrolyte, arid the tip immersed in a phosphate- freeTris-HCl buffer. An aliquot of zinc chloride solution was added to thebath and stirred by repeated pipetting. The system was monitored whileapplying voltages from +500 to −800 mV. Experiments at different pHvaried the phosphate buffer in the barrel only, with pH values of 6, 7,8, or 10.

Kinetics of Current Oscillations

Nanopipettes were selected with a current value of −3500 to −4500 pA ata potential of −500 mV. Potentials from −300 to −500 mV produced currentoscillations, for which a threshold was set for high and low conductancestates. By measuring the time from high to low conductance, the slope ofpore closing was estimated in pA per ms. The high and low conductancestates were set as follows: −500 mV, −1700 and −1200 pa; −400 mV, −1200and −700 pA; −350 mV, −900 and −400 pA; −300 mV, −1000 and −500 pA. Fortemporary opening of the pore, a biphasic waveform was used, where theapplied potential oscillated between +500 and −500 mV for a period of2.5 s each. The threshold for high conductance was set at −3500 pA, andthe low conductance state at −2000 pA. The slope was calculated from thetime required to reach the low conductance state.

Data Analysis

Data was sampled at a rate of 1 kHz using Clampex software. Dataprocessing was done using Clampfit and OriginPro 8.5 (OriginLab,Northhampton, Mass.). Calculation of relative time in high vs. lowconductance states used the peak finding function of OriginPro to findeither negative (high conductance) or positive (low conductance) peaks,and the number of events in each state calculated as a percentage of thetotal events.

Results

To use the nanopipette as a nanoreactor, conditions to control theprecipitation of zinc phosphate at the pore through ion migration wasestablished. In a typical setup, an Ag/AgCl electrode is inserted intoan electrolyte solution (100 mM KCl with 10 mM buffer) that fills thebarrel of a nanopipette. The pipette tip is immersed in an electrolytebath, which also contains an Ag/AgCl ground electrode (see FIG. 1).

On applying a potential, a steady ion current is measured. However, onadding micromolar concentrations of zinc chloride to the bath, thesystem undergoes oscillating periods of high and low conductance. Thesecycles were attributed to precipitation of highly insoluble zincphosphate inside the nanopipette tip, and its subsequent evacuation fromthe pore. The oscillations caused by nanoprecipitation in such pipettesare on the order of seconds, as shown in FIG. 3, and are marked by afluctuating state of low conductance and rapid, short-lived oscillationsto a state of high conductance. The time plot of (FIG. 3) of the ioncurrent at −350 mV potential shows several precipitation events that donot terminate with complete opening of the pore; rather, there are manyevents in which the low-conductance state fluctuates between −400 and−700 pA. These are likely due to precipitates that are evacuated beforethey grow to a size sufficient to completely block the pore. When astate of high conductance is reached, however, the current consistentlyreaches a maximum value of roughly −1200 pA followed by a rapid drop to−400 pA. This indicates a complete clearing of the pore followed byrapid precipitation.

To show that the precipitation reaction is controlled by thevoltage-induced ion migration of zinc and phosphate ions, and does notsimply occur by mixing at the nanopore, which is the interface of thetwo solutions, the zinc and phosphate counter ions were isolated in twoseparate solutions; phosphate ions are confined to the inside of thenanopipette, and zinc ions are in the bath (FIG. 1), The minimumrequired voltage to induce oscillating current blockage in this systemis −300 mV. At potentials from +500mV to −200 mV, a stable current isseen. At −300 mV, the current immediately becomes blocked with rapidfluctuations. The existence of a voltage threshold for thenanoprecipitation reaction indicates there is little mixing between thetwo counter-ions at the interface of the two solutions. Applying apositive potential gives a signal of smaller magnitude due to currentrectification, but the signal is not influenced by the presence of zincor phosphate salts. This voltage-dependent effect is consistent with themovement of ions toward the electrode of opposite charge, with phosphateand zinc ions meeting at the pipette tip (FIG. 2). When the placement ofsolutions is reversed such that zinc chloride is inside the pipettebarrel and phosphate is in the bath, no blockage occurs with either apositive potential or a negative potential. While a positive potentialin this configuration can in theory cause precipitation as zinc ions arepushed out of the pipette and phosphate migrates into the pore from thebath, this is not observed. The lack of current blockage in thisconfiguration may be due to exclusion of cations (such as zinc) at theinner tip of the pipette, a phenomenon often cited as a cause of currentrectification in conical nanopores.

Further investigation was conducted on the nature of the precipitate byvarying the pH and the concentration and composition of the ions in thebath. It was assumed that the precipitate is composed of zinc phosphate,a salt which is highly insoluble in aqueous systems with K_(sp) of 10⁻³⁵(mol.L⁻¹). While the predominant species in solution at pH 7 aredihydrogen phosphate (H₂PO₄) and hydrogen phosphate (HPO₄ ²⁻), zincphosphate is thermodynamically stable and forms in solutions at neutralor acidic pH. A solution is saturated with roughly 1×10⁻⁷ M phosphateand zinc ions. Oscillating current behavior was seen in pipettes filledwith phosphate buffer from pH 6 to pH 10, and with zinc chloride addedto the bath at concentrations between 2 and 40 μM. While other divalentions such as calcium and magnesium were tested at those concentrations,the only other comparable blockage was with iron(III) chloride (10 μM),which irreversibly blocked the pore. This is likely due to iron(III)hydroxide precipitation, a salt even less soluble than zinc phosphate(K_(sp) Fe(OH)₃ 10⁻³⁹).

There are several possible mechanisms by which a nanopore blocked byprecipitation spontaneously becomes cleared. For current oscillationsseen with phosphate salts of calcium and cobalt in PET track-etchednanopores, the precipitation was attributed to voltage-inducedconcentration of salts in an asymmetric nanopore, causing a localincrease in salt concentration to supersaturation levels. This led to ahypothesis in which the precipitate rapidly dissolves due to iondepletion in the nanopore. A computational study supported a secondmechanism, wherein protons donated by hydrogen phosphates in theprecipitate are accepted by oxides at the pore surface, weakening thepore-particle interaction and allowing the particle to clear bymigration. For the pore blockage reported here, the effect is onlyobserved at concentrations between 1 and 100 micromolar zinc chloride,well above the saturation level for zinc phosphate. Thus, the lattermechanism offers an explanation for the oscillations observed here. Tosupport this mechanism, current oscillations were induced in a pipettefilled with phosphate buffer and immersed in a saturated solution ofzinc phosphate. The precipitate in such a case is likely ejected fromthe pore, rather than dissolved (see FIG. 14). With a negativepotential, shown in FIG. 14 as V<0 in the bore, oppositely charged ionsmigrate to the interface of the solutions inside and outside thepipette. Zinc phosphate precipitates at the pore, causing ion current todecrease. When the precipitate has grown to sufficient size, it iscleared from the pore by electrophoretic forces.

If the zinc phosphate precipitate migrates out from the pore asbelieved, it remains to be explained why current oscillations are seenonly with negative potentials. While the exact chemical composition ofthe precipitate is unknown at this time, clusters of zinc phosphate havebeen shown to have a net negative charge at neutral pH, as measured byzeta potential. Precipitation and blocking of the pore will lead to anincreased electric field at the pore, and thus a negative potential maymove the precipitate out of the pore and into the bath byelectrophoretic and electroosmotic forces. While the applied voltagesare low in these experiments (−300 to −500 mV), the voltage drop will begreatest across the region of highest impedance, such as the blockedpore. Ejection of the particle by electrophoretic forces may also helpto explain why iron(III)hydroxide does not exhibit spontaneous clearingfrom the pore, as the particles carry a positive charge and would beexpected to have a strong interaction with the negatively charged quartzsurface. Interestingly, a pipette showing positive current rectificationafter deposition of a poly-L-lysine electrolyte layer became blockedfrom voltage-induced mixing of zinc phosphate, but did not display anyoscillations to an open state. Presumably, the negatively chargedprecipitate has a high affinity for a positively charged pore and cannotbe dislodged as easily.

In addition to initiating the process of nanoprecipitation with thevoltage-controlled nanoreactor, the size of the precipitate may also becontrolled. If the pore is cleared due to electrical forces at the tipof the nanopipette, then increasing the potential is expected to ejectsmaller particles that have not completely blocked the pore. This wasdemonstrated experimentally on reaching a potential of −600 mV, wheretwo distinct low-conductance states are seen (FIG. 15).

A histogram showing counts for different current levels shows a state ofhigh and low conductance for −300, −400, −500, and −600 mV. At −600 mV,there is clearly more time spent in the open state relative to theclosed state, also visible in the time plot (FIG. 15A). For example, thesystem at −300 mV shows 41% time spent in a high conductance state,while at −600 mV, that value is 73%. This indicates that as thepotential increases, the precipitate is prevented from blocking thepore. Unlike the other voltages measured, the time plot at −600 mV showsthree states: a low conductance state (−800 pA) that occurs infrequentlyand for longer duration than an intermediate state (−3000 to −3500 pA),and a high conductance state (−6000 pA). It is believed that theintermediate state corresponds to precipitates that are ejected afteronly partially blocking the pore. At voltages less than −600 mV, theprecipitate is cleared only after it has grown to sufficient size tocompletely block the pore.

It is expected that at some point salt will accumulate in the pore to anextent that the precipitate cannot be ejected. This stage ofprecipitation was also studied with the nanoreactor, and revealed anunforeseen phenomenon. Many of the nanopipettes underwent three stagesof blocking by nanoprecipitation. The first stage was that ofspontaneous current oscillations with a constant negative appliedpotential. After a period of 20 minutes or more, the pore became blockedand exhibited a steady state of low-conductance. At this stage, however,the pore could be temporarily forced into a high-conductance state by arapid pulse of positive potential. Finally, the pipette would becomeirreversibly blocked. For the first two stages, the goal was tounderstand what was occurring in the pipette as the pores are becomingcleared and subsequently blocked. The kinetics of pore opening cannot becompared for the two systems, as they occur under potentials of oppositepolarity. The kinetics of pore closing was investigated to find if thereare different mechanisms at work for a pipette in the two stagesdescribed.

If the pore can be cleared by a negative potential causing migration ofa negatively charged particle out through the nanopore, then a positivepotential is expected to move the precipitate in the opposite direction,to the broader shaft of the pipette tip. For blocked pores, a pulse of+500 mV was briefly applied (0.2 to 2 s), followed by reversal of thevoltage to −500 mV. At the negative voltage, a high conductance state isseen from the previously blocked pore, which again becomes rapidlyblocked (data not shown). The brief open state is of the same magnitudeas the open states in pipettes undergoing current oscillations, and istherefore attributed to an open pore rather than a transient current dueto the rapid change in voltage. The temporary high- conductance stateindicates that the precipitate has migrated away from the pipette tipand is either replaced by precipitation from solution, or that theparticle is moved back toward the pore when the potential is reversedfrom positive to negative.

The kinetics of pore closing will be distinct for the two differentmechanisms, nanoprecipitation vs. migration of a particle into the pore.To compare blocking kinetics in oscillating vs. blocked pores, the rateof current blockage for individual events at −500 mV for both conditionswas quantified. For blocked pores that have been briefly opened with a+500 mV pulse, the current at a blocking event decreases with a slope of74±13 pA/ms, as compared to 4±2 pA/ms for a pore undergoing currentoscillations. The significantly faster current blockage indicates ablocking mechanism other than nanoprecipitation from solution. Rather,this may represent voltage-driven shuttling inside the nanopipette, frombase to tip, of a particle too large to exit through the nanopore. Thisphenomenon has thus far only been observed with zinc phosphate salts,and did not occur with blockage from other precipitates such as iron(III) hydroxide.

If the precipitate in a blocked nanopipette is indeed moved within thetip during pulses of positive potential, then the particle can betrapped with alternating voltages to leave the pore unblocked. Byapplying sinusoidal potentials of sufficiently high frequency, an “open”current can be produced from pipettes which appear to be blocked by zincphosphate. For measurement, a pipette filled with pH 7 phosphate buffer(10 mM with 100 mM KCl) was immersed into a bath of pH 7 Tris buffer (10mM with 100 mM KCl) containing 2μM zinc chloride. The pipette becameblocked after constant potential of −500 mV for several minutes, andsinusoidal potential from 500 to −500 mV was applied at frequencies of0.1, 0.5, 1, and 5 Hz. At lower frequencies such as 0.1 Hz, the pore canbe seen to briefly approach a high-conductance state before becomingblocked as the potential oscillates to a negative voltage. At higherfrequencies, a much higher conductance state is achieved, and the poreis cleared by a positive potential before it becomes blocked.

The high-conductance state achieved with higher frequencies ofsinusoidal voltage represents trapping in space of a nanoprecipitateusing an oscillating electric field, and also allows the magnitude ofcurrent to be precisely controlled with frequency of the appliedpotential.

Example 8 Carbohydrate (Saccharide)-Responsive Functionalized PolymerCoating

Nanopipette sensors were developed for sensing saccharides in a samplesolution using boronic acid chemistry applied to the nanopore channel.The rationale is that while saccharides may be small relative to thepore (which may be e.g. 10−40 nm in opening diameter), the binding ofsaccharides to boronic acids can cause the neutral boronic acid toconvert to the negatively charged boronate. Nanopipettes and otherconical nanopores exhibit current rectification, or ion perselectivity,which is sensitive to surface charge. Initial attempts to makereversible sensors based on the known methods of surfacemodification—either through covalent attachment of boronic acids (aschematic of which is illustrated in FIG. 17) or deposition of afunctionalized polyelectrolyte—resulted in sensors that were either notsufficiently sensitive or responded irreversibly. In a nanopipettecovalently modified as shown in FIG. 17, the ion current responded to 3mM glucose in pH 7 buffer. It was reasoned that the sensitivity of thenanopore would be increased if the recognition element covered theentire cross section of the pore, and not only the pore walls (see FIG.18C for a diagrammatic representation of a polymer crosslinked so as toextend into and/or across the nanopore; note that the polymer is in amesh-like state so as to partially block, but not to completely blockthe nanopore; the mesh will generally be at the most distal portion ofthe interior of the nanopore). The potential sensitivity of such asystem has been demonstrated with covalently attached, pH-responsive“polymer brushes,” which changed rectification behavior of thin-filmnanopores as a result of protonation of phosphate groups on the polymer(Yameen, et al. Chem. Commun. 46, 1908−1910 (2010). Such a system,however, relies on a charged analyte (hydronium ion) to elicit aresponse. Noprevious nanopore sensors are known in which binding of aneutral analyte causes a change in charge state of the nanoporeenvironment, influencing current rectification.

Methods

Reagents and Solutions: All stock solutions were prepared in Milli-Qultrapure water. Buffer solutions were prepared from potassium chloride(Baker), sodium phosphate (dibasic), sodium carbonate, and sodiumbicarbonate (Sigma), and adjusted with either HCl (1 M) or KOH (0.1 M).Alizarin red sulfonate (ARS), 8-Hydroxypyrene-1,3,6-trisulfonic acid,trisodium salt (HPTS), esculetin, L-glucose, and L-fructose werepurchased from Sigma. All buffer solutions used for analysis contained10 mM buffer and 100 mM potassium chloride unless otherwise indicated.

Synthesis of Polymer PVP-BA: Poly(4-vinylpyridine) (MW 60,000) waspurchased from Sigma and used as received. The synthesis ofo-bromomethylphenylboronic acid was carried out using an establishedprocedure. To a 10 mL round bottom flask containing a magnetic stir barwere added poly(4-vinylpyridine) (0.100 g, 0.00167 mmols) andm-bromomethyl phenylboronic acid (0.206 g, 0.954 mmols). ThenN,N-dimethylformamide (2 mL) and methanol (2 mL) were added to dissolvethe reagents. The mixture stirred 23 hours, then was added dropwise to a50 mL beaker containing dichloromethane (10 mL) to precipitate theproduct. The beaker was placed in an ice bath to allow the completeprecipitation of the product. The solution was then poured into atwo-piece fritted filter with removable top and vacuum-filtered underinert conditions using argon gas. The product was washed with 3×15 mLportions of dichloromethane, then left in a vacuum dessicator to dryovernight. Product isolated was 0.257 g (90% yield). ¹H-NMR showed 82%alkylation of the polymer (data not shown).

Measuring Carbohydrate Response with Fluorimetry: A probe solution wasprepared consisting of PVP-BA (0.006% w/v) and HPTS (1.5×10⁻⁶ M) inmethanol/water (1:1). An aliquot of 1 mL of the probe solution was addedto a cuvette followed by 1 mL of carbonate buffer (20 mM carbonate, 100mM potassium chloride, pH 9.5). The fluorescence was read using a filterpair of 460 nm (excitation) and 515−570 nm (emission). Aliquots ofsaccharide solutions (500 mM in water) were added, the solution wasmixed by slowly pipetting for 1 min, and the fluorescence was measuredafter each addition. The total volume added did not exceed 20microliters (1% of total volume). The fluorescent signal was convertedto fluorescence increase (F/F₀). The averaged signal from multiple blankvalues was taken as the baseline fluorescence, F. Dividing each signalby this blank value gave the fluorescence increase.

Current Measurement with Quartz Nanopipette Electrodes: Nanopipetteswere fabricated using a P-2000 laser puller (Sutter Instrument Co.) fromquartz capillaries with filaments, with an outer diameter of 1.0 mm andan inner diameter of 0.70 mm (QF100−70−5; Sutter Instrument Co.).Parameters used were: Heat 625, Filament 4, Velocity 60, Delay 170, andPull 180. To measure ion current, a two electrode setup was used. Thenanopipette was backfilled with buffer solution (phosphate/KCl, pH 7)and an Ag/AgCl electrode inserted. Another Ag/AgCl electrode was placedin 0.3 mL bulk solution acting as auxiliary/reference electrode. Bothelectrodes were connected to an Axopatch 700B amplifier with theDigiData 1322A digitizer (Molecular Devices), and a PC equipped withpClamp 10 software (Molecular Devices). To ensure complete wetting ofthe nanopipette electrodes, nanopipette tips were immersed inN,N-dimethylformamide for 5-10 seconds after being backfilled withbuffer. Positive potential refers to anodic potential applied to theelectrode in the barrel of the nanopipette relative to the counterelectrode. Experiments were carried out at 24° C.

Embedding PVPBA in Nanopipettes: Nanopipette barrels were filled withphosphate buffer (pH 7) and immersed in carbonate buffer (pH 9.5)containing the counter electrode. After verifying the nanopipettesdisplayed negative current rectification, they were briefly immersed ina methanol solution containing 0.03% (w/v) polymer, then returned to thecarbonate solution. Successful immobilization of the polymer resulted incomplete reversal of current rectification.

Measuring Carbohydrate Response with Polymer-Modified Nanopipettes:Modified nanopipettes were analyzed in 0.30 mL of a carbonate buffersolution (pH 9.5) containing the counter electrode. To the solution wereadded aliquots of concentrated analyte solutions in pure water. Thetotal volume added did not exceed 15 μL, in order to limit the change involume to 5%. To measure response in real time, the current was analyzedusing a sinusoidal potential at frequency of 0.5 Hz from −500 to +500mV. After the signal had stabilized following addition of an aliquot,the current was analyzed by sweeping the voltage from −500 to +500 mV ata rate of 0.5 mV/ms. Each measurement consisted of 5 sweeps.

Electrochemical Data Analysis: Ion current measurements recorded withpClamp software (sampling frequency 1000 Hz for voltage sweeps, 200 Hzfor sinusoidal function) were imported to OriginPro 8.5 (Origin Labs)for analysis and graphing. To generate I-V curves for each data point, 5voltage sweeps from −800 to +800 mV at a scan rate of 500 mV s⁻¹ wereaveraged and the standard deviation calculated for each point. Togenerate binding isotherms, the current at a fixed potential was plottedas a function of analyte concentration.

Chemical Properties of the Functional Polyelectrolyte

To make a cationic, carbohydrate-responsive polymer, m-bromomethylphenylboronic acid was used to alkylate a commercially availablepoly(4-vinylpyridine) (PVP) of molecular weight 60,000. The product,PVP-BA, precipitated from the reaction mixture in N,N-dimethylformamideand methanol. It is weakly soluble in methanol (up to 1% w/v), sparinglysoluble in acidic methanol/water solutions, and practically insoluble inother aqueous and organic solvents. A benzylated version of the polymer(PVP-Bn), alkylated with benzyl bromide, showed much higher solubility.The alkylation efficiency of the polymers was determined by integrationof ¹H-NMR spectra. Polymer PVP-BA showed approximately 85% alkylation,while for PVP-Bn the alkylation efficiency was 90%. These values wereused to estimate the molar mass of the polymers as 170,000 for PVP-BAand 150,000 for PVP-Bn. Polyelectrolytes such as poly(vinylpyridine) canbe analyzed by titration to characterize protein uptake and apparentpK_(a).³²

To evaluate the pH-dependence of polymer PVP-BA, a solution of 1% w/v inmethanol/water was acidified to pH 2. When titrated with hydroxide, thepolymer quickly precipitated at approximately pH 8. This precipitationpoint was highly reproducible, and is consistent with the pK_(a) ofphenylboronic acid in solution. At that point, conversion of the boronicacid to boronate will effectively neutralize the charge from pyridiniumgroups, forming a zwitterionic polymer that is much less polar. Thisprecipitation point is modulated in the presence of 20 mMmonosaccharides (see pH at precipitation data, FIG. 20), which are knownto lower the pK_(a) of boronic acid by as much as 2 pH units. Comparedto the precipitation of PVP-BA at pH 7.8±0.2, glucose causes theprecipitation to occur at pH 6.5±0.1, and fructose causes precipitationat pH 5.58±0.08. Fructose is known to have a high affinity with mostboronic acid receptors. This work shows that both glucose and fructosebind to the PVP-BA used. This method can be used to determine whethervarious polyelectrolytes can be used as coatings on the nanopipette, andto adjust the pH responsiveness of the polymers, e.g. by addingnegatively chareged groups or altering the positions of the negativelycharged groups.

To better characterize the interaction of the cationic polymer withcarbohydrates, three colorimetric dyes were selected that can eachinteract with the polymers PVP-BA and PVP-Bn. These dyes act assurrogates for glucose sensing, were commercially available, and havepublished structures. Alizarin red sulfonate (ARS), contains both acatechol and a negative charge, esculetin, which contains only acatechol, and 8-Hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt(HPTS), a dye with three negative charges. Solutions of each dye weretitrated with polymer, and the absorbance spectra recorded. Arepresentative absorbance spectrum is shown in FIG. 21 for ARS dyetogether with either PVP-BA or PVP-Bn. In the presence of PVP-Bn, whichdoes not contain boronic acids, the absorbance maximum at 420 nmdecreases and is slightly red-shifted to 432 nm. When PVP-BA is added tothe dye, the maximum absorbance increases and is shifted to 467 nm.These two distinct phenomena show an interaction due to both boronicacid binding and electrostatic interactions. Also, the ARS bound to theBA polymer, but not the Bn polymer.

By measuring the difference in absorbance at λ_(max), a curve wasobtained showing AA as a function of polymer concentration. Thesensitivity of each dye to the polymers was measured using the slope ofthe linear portion of curve, summarized in Table 1. For PVP-BA, theboronic acid-appended polycation, the affinity for ARS was double thatof HPTS, showing a synergistic effect of both electrostatic attractionand bond formation. In contrast, PVP-Bn showed stronger affinity forHPTS compared to ARS, consistent with an all-electrostatic mechanism.Significantly, the affinity for esculetin is one order of magnitudelower for PVP-Bn than PVP-BA. This shows that without the presence ofthe boronic acid, there is little interaction between the polycation anduncharged catechol. While plotting M vs. polymer concentration produceda smooth curve for all dyes tested, none showed a good fit to a standardbinding isotherm. This is likely due to the complex nature of thepolymer, which has multiple binding sites (approximately 500 per polymerchain) and may undergo aggregation in the presence of the dyes.

TABLE 1 Relative affinity of polymers for colorimetric dyes. Sensitivity(μM⁻¹) PVP-BA PVP-Bn ARS 2.09 ± 0.04 0.70 ± 0.02 Esculetin 1.53 ± 0.050.11 ± 01   HPTS 1.02 ± 0.05 1.53 ± 0.02 Note: Dyes were titrated withsolutions of the polymers, and the slope was calculated as change inλ_(max) as a function of polymer concentration.Nanopipettes Embedded with Functional Polymer

To form a nanochannel functionalized with the boronic acid-containingpolycation PVP-BA, quartz nanopipettes (pore diameter 20-40 nm) filledwith phosphate buffer were used. In this medium, the polymer isinsoluble. Before addition of the polymer, these show negativelyrectified ion current at pH 7. That is, the IV plot shows higher currentat negative voltage. To functionalize the nanochannel with the polymer,nanopipettes were briefly immersed in a methanol solution containing thepolymer at 0.3% concentration (w/v). On returning the nanopipette tip toneutral buffer solution the current rectification is reversed, showingnonlinear conductance that is higher at positive potentials. The PVP-BApipette showed greater ion current at +voltage, and very little currentat negative potential.

The inverted current rectification is evidence that the polymerpenetrates the pore of the nanopipette, where the impedance of thesystem is highest. Several such polymer-modified nanochannels wereproduced in which the positive rectification was stable over a matter ofhours. No polymer was visible on the outside of the nanopipette.Presumably, the modified ion current arises from polymer that isembedded in the nanochannel, held in place by both electrostaticattraction to the negatively charged quartz and limited solubility inthe buffer. Imaging of the polymer within the nanochannel is notpractical, but to approximate the system a micropipette was subjected tosimilar treatment. A micropipette of 20 micrometer pore diameter wasfilled with phosphate buffer containing 1 mM ARS for visualization ofthe polymer. Immersion in a methanol solution of the polymer produces aviolet color at the very tip of the micropipette. Even after 20 minutes,very little of the polymer diffused up into the wider opening of thepipette tip.

The pH sensitivity of modified nanopipette electrodes is considerablygreater than quartz nanopipettes. The negative rectification of a barequartz nanopipette shows only a slight decrease at pH 3, correspondingto protonation of silanoxy groups. In contrast, a nanopipette withembedded PVP-BA shows virtually no conductance at negative potentialsfrom pH 8 to pH 3 (data not shown). The conductance at positivepotentials increases with decreasing pH, showing the most change betweenpH 5 and pH 3. The electrochemical behavior of the nanochannel/polymermaterial is not predicted from the properties of the polymer insolution, which undergoes the biggest change in protonation statebetween pH 7 and 8.

The anionic catechol ARS showed the highest affinity for polymer PVP-BAin solution among the dyes tested. To test modulation of ionpermselectivity with this dye, polymer-modified nanopipettes wereimmersed in carbonate buffer of pH 9.5. Under these basic conditions,the formation of anionic boronate esters is ensured.³⁴ As shown in FIG.22, as little as 60 μM ARS is sufficient to negate all positiverectification in a modified nanochannel. With 360 μM ARS, the current isnegatively rectified. Referring to FIG. 22, the arrow at 360 shows theincreased negative current at a negative voltage. In this example, anegative current of about −1 nA is observed at −500 mV, while +500 mVresulted in about +0.6 nA. The example also shows how the blank, 60 μMand 360 μM concentrations of ARS can be distinguished. Because the ioncurrent rectification becomes reversed, addition of the dye does notappear to cause blockage of the nanopore, or nanochannel. Rather, theion permeability of the channel is reversed based on reversal ofelectrostatic charge within the matrix.

At low concentrations of ARS (<0.1 mM), the modulation of ion currentrectification is completely reversible, requiring no washing media.Higher concentrations caused the system to become permanently negativelyrectified. This may be due to strong interactions between the polymerand the dye, especially if the dye penetrates deep into the polymermatrix where it is prevented from diffusing into the bulk solution.

The reversal of ion current rectification in the modified nanochannelshown with ARS may be due to both the charge of the dye as well as apK_(a) shift in the boronic acid-containing matrix. For neutralcarbohydrates, a change in rectification must be attributed to boronicacid/boronate equillibria in the nanochannel. The addition of fructose(10 mM) to a polymer-embedded nanochannel resulted in rapid inversion ofcurrent rectification from positive to negative (FIG. 23-24). The curvesin FIG. 23 show that there is almost no rectification in the blanknanopipettes, while the presence of fructose at 10 mM resulted in an IVcurve exhibiting negative rectification (i.e. higher current at negativevoltage). FIG. 24 shows the reversibility of the binding, in that theresponse time for complete inversion of current rectification is 3 to 5minutes, both on exposure to fructose and on returning the nanopipettetip to pure buffer. Interestingly, only a portion of the initialpositively rectified I-V curve is restored after exposure to fructose.As shown in FIG. 23, the conductance at negative potentials undergoescomplete and reproducible switching in repeated cycles of fructoseexposure. This is not necessarily the case for positive potentials. Asshown in FIG. 23, the initial signal for a PVP-BA modified nanopipetteis highly open at positive potentials up to 600 mV. This open state doesnot become completely restored after exposure to fructose. Thisindicates some conditioning of the matrix in the nanochannel as a resultof carbohydrate binding. While the magnitude of current rectificationdiffered among different polymer-embedded nanochannels, the inversion ofrectification in the presence of fructose was complete and reversiblefor several systems tested (FIG. 25). Exemplary spots 254 and 255 showpositive and negative rectification, respectively, in differentexperiments.

Because of the complete reversal of current response at negativepotentials, a current at fixed negative potential can be used to showfructose modulated gating between an open and closed state. As shown inFIG. 24, the current at −500 mV is completely reversible with severalcycles of fructose exposure. Significantly, there are no washingconditions required to restore the signal. It should also be noted fromthe smoothness of the I-V curves that the polymer matrix does not appearto be influenced by the electric field. Rather, it is only the presenceof a neutral carbohydrate that modulates ion permeability in thenanochannel.

The response of the modified nanochannel to fructose, as with ARS, isconcentration-dependent (FIGS. 26-27). By plotting the current atpotential of −500 mV as a function of fructose concentration, a bindingaffinity can be determined for a given nanochannel, given by thefollowing equation:

S=(1+S _(max) K _(b) [A])/(1+K _(b) [A])

This model uses S as the signal, in this case ion current, S_(max) asthe calculated signal upon saturation with analyte, [A] as the analyteconcentration, and K_(b) as the binding constant in units of M⁻¹. Thebinding constant determined from fitting of the curve is 360±110 M⁻¹ forthe nanochannel shown, which is consistent with binding constantsmeasured using mono-phenylboronic acids in solution. These affinitiescan vary widely, but are generally within the range of 100 to 5000 M⁻¹.To compare the response of the nanochannel-confined polymer to thesolution phase polymer, a fluorescence-based assay was carried outwhich, like the nanochannel electrode, is modulated by electrostaticcharge. Using the dye HPTS, an allosteric indicator-displacement assay(AIDA) was used to measure fructose response. In solution, HPTS forms aground-state complex with PVP-BA polymer, quenching fluorescence. Thetwo components are attracted electrostatically; the cationicpolyelectrolyte to the anionic dye. At pH 9.5, the addition of fructosecauses boronic acid groups on the polymer to become anionic boronates,neutralizing the overall positive charge on the polymer and displacingthe fluorescent dye. With this system, an apparent binding constant wasdetermined as 3200±400 M⁻¹ for fructose (FIG. 26). This affinity is anorder of magnitude greater than that measured with the polymer in aconical nanochannel, but still well within reported values for boronicacid-fructose binding. While the environments for the polymer are verydifferent for both systems, in both cases it is a modulation in polymercharge that gives a signal. The ability to quickly engineer astimulus-responsive nanofluidic diode using a well-characterizedreceptor matrix offers a new strategy to control ion permeability innanopores.

Thus the data in FIGS. 25 and 26 show that the fluorescence valuesobtained in an accepted assay are comparable to the ion current valuesobtained at varying concentrations of fructose at a set negativevoltage, and that the present electrochemical method has a higherdynamic range than the fluorescence method.

Sensors comprising nanopipettes embedded with PVP-BA polymer were alsoshown to respond to glucose with an increase in negative rectificationat pH 9.5. A sensor showing negative rectification ratio of 1.42 inbuffer was exposed to 20 mM glucose, whereupon the negative currentincreased resulting in negative rectification ratio of 1.99. Negativecurrent ratio is defined as the ratio of current at potential −500 mV tothe current at potential +500 mV.

Conclusion

The above specific description is meant to exemplify and illustrate theinvention and should not be seen as limiting the scope of the invention,which is defined by the literal and equivalent scope of the appendedclaims. Any patents or publications mentioned in this specification areintended to convey details of methods and materials useful in carryingout certain aspects of the invention which may not be explicitly set outbut which would be understood by workers in the field. Such patents orpublications are hereby incorporated by reference to the same extent asif each was specifically and individually incorporated by reference andcontained herein, as needed for the purpose of describing and enablingthe method or material referred to.

1-29. (canceled)
 30. A nanopipette for measuring pH of a sample, thenanopipette comprising: (a) a capillary portion defining an interiorbore of the nanopipette leading to a nanopore; (b) the interior boreadapted for containing therein an electrode and an interior solutioncommunicating with an exterior solution through said nanopore; and (c) acoating on an interior surface of the nanopore, the coating comprising:(i) a polyelectrolyte layer bound directly to the interior surface; and(ii) a chitosan molecule linked to the polyelectrolyte layer.
 31. Thenanopipette of claim 30, wherein said polyelectrolyte is a polycation.32. The nanopipette of claim 31, wherein said polycation is a polyalkylpyridine or a polyamine.
 33. The nanopipette of claim 31, wherein thepolyelectrolyte layer is selected from the group consisting of (a) apolyacrylic layer, (b) a polyamine layer; and (c) alternating layers ofpolyacrylic and polyamine.
 34. The nanopipette of claim 33, wherein thepolyamine is a polyalkyl pyridine.
 35. The nanopipette of claim 30,wherein the nanopipette is a quartz nanopipette.
 36. A nanopipetteapparatus for measuring pH of a sample, the apparatus comprising: (a) ananopipette having an interior bore and a nanopore opening into thesample; (b) an electrode within the interior bore, arranged to contactan interior solution, and a reference electrode positionable in thesample; (c) a coating on an inner surface of the nanopore; (d) thecoating comprising: (i) a polyelectrolyte layer bound directly to theinterior surface; and (ii) a chitosan molecule linked to thepolyelectrolyte layer; and (e) a voltage control circuit for generatinga voltage between electrodes and measuring ionic current through thesample, the nanopore, and the interior solution.
 37. The nanopipetteapparatus of claim 36, wherein the nanopipette is quartz.
 38. Thenanopipette apparatus of claim 36, wherein the coating comprises apolycation.
 39. The nanopipette apparatus of claim 36, wherein thepolycation is a polyalkyl pyridine or a polyamine.
 40. The nanopipetteapparatus of claim 36, wherein the polyelectrolyte layer is selectedfrom the group consisting of (a) a polyacrylic layer, (b) a polyaminelayer; and (c) alternating layers of polyacrylic and polyamine.
 41. Thenanopipette of claim 40, wherein the polyamine is a polyalkyl pyridine.42. A method for measuring pH of a sample, the method comprising:contacting the sample with the nanopipette apparatus of claim 36; andusing the voltage control circuit for measuring ionic current, whereinthe current is correlated to pH of the sample.
 43. The method of claim42, wherein the nanopipette is quartz.
 44. The method of claim 42,wherein said coating comprises a polycation.
 45. The method of claim 44,wherein the polycation is a polyalkyl pyridine or a polyamine.
 46. Themethod of claim 42, wherein the polyelectrolyte layer is selected fromthe group consisting of (a) a polyacrylic layer, (b) a polyamine layer;and (c) alternating layers of polyacrylic and polyamine.
 47. The methodof claim 46, wherein the polyamine is a polyalkyl pyridine.
 48. Themethod of claim 42, wherein the sample comprises a single cell and themethod measures pH of the cell.